J Pediatr (Rio J).

2018;94(5):554---558

www.jped.com.br

ORIGINAL ARTICLE

Human parainfluenza virus surveillance in pediatric

patients with lower respiratory tract infections: a
special view of parainfluenza type 4夽
Luciano M. Thomazelli a,∗ , Danielle B. L. de Oliveira a , Giuliana S. Durigon b ,
Brett Whitaker c , Shifaq Kamili c , Eitan N. Berezin b , Edison L. Durigon a

a
Universidade de São Paulo (USP), Instituto de Ciências Biomédicas, São Paulo, SP, Brazil
b
Irmandade da Santa Casa de Misericórdia de São Paulo, São Paulo, SP, Brazil
c
Center for Disease Control and Prevention, Atlanta, United States

Received 13 April 2017; accepted 11 July 2017

Available online 28 September 2017

KEYWORDS Abstract
Human parainfluenza Objective: Characterize the role of human parainfluenza virus and its clinical features in Brazil-
virus; ian children under 2 years of age presenting with acute lower respiratory tract infections.
Respiratory virus; Methods: Real-time assays were used to identify strains of human parainfluenza virus and other
Pediatric patients; common respiratory viruses in nasopharyngeal aspirates. One thousand and two children pre-
Acute respiratory senting with acute lower respiratory tract illnesses were enrolled from February 2008 to August
illness; 2010.
Human respirovirus 4 Results: One hundred and four (10.4%) patients were human parainfluenza virus positive,
of whom 60 (57.7%) were positive for human parainfluenza virus-3, 30 (28.8%) for human
parainfluenza virus-4, 12 (11.5%) for human parainfluenza virus-1, and two (1.9%) for human
parainfluenza virus-2. Seven (6.7%) patients had more than one strain of human parainfluenza
virus detected. The most frequent symptoms were tachypnea and cough, similar to other viral
respiratory infections. Clinical manifestations did not differ significantly between human parain-
fluenza virus-1, -2, -3, and -4 infections. Human parainfluenza virus-1, -3, and -4 were present
in the population studied throughout the three years of surveillance, with human parainfluenza
virus-3 being the predominant type identified in the first two years.

夽 Please cite this article as: Thomazelli LM, Oliveira DB, Durigon GS, Whitaker B, Kamili S, Berezin EN, et al. Human parainfluenza

virus surveillance in pediatric patients with lower respiratory tract infections: a special view of parainfluenza type 4. J Pediatr (Rio J).
2018;94:554---8.
∗ Corresponding author.

E-mail: lucmt@usp.br (L.M. Thomazelli).

https://doi.org/10.1016/j.jped.2017.07.017
0021-7557/© 2017 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Human parainfluenza virus in pediatric patients 555

Conclusion: Human parainfluenza viruses contribute substantially to pediatric acute respiratory

illness (ARI) in Brazil, with nearly 30% of this contribution attributable to human parainfluenza
virus-4.
© 2017 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. This is an open
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
4.0/).

PALAVRAS-CHAVE Vigilância dos vírus parainfluenza humanos em pacientes pediátricos com infecções
Vírus da parainfluenza do trato respiratório inferior: uma visão especial do parainfluenza tipo 4
humana;
Resumo
Vírus respiratório;
Objetivo: Caracterizar o papel do VPH-4 e suas características clínicas em crianças brasileiras
Pacientes
com menos de dois anos de idade com infecções agudas do trato respiratório inferior.
pediátricos;
Métodos: Ensaios em tempo real foram utilizados para identificar tipos de VPH e outros vírus
Doença respiratória
respiratórios comuns em aspirados nasofaríngeos. Mil e duas crianças com doença aguda do trato
aguda;
respiratório inferior foram inscritas para participar de fevereiro de 2008 a agosto de 2010.
Respirovírus humano
Resultados: 104 (10,4%) pacientes eram VPH positivos, dos quais 60 (57,7%) eram positivos
4
para VPH-3, 30 (28,8%) para VPH-4, 12 (11,5%) para VPH-1 e dois (1,9%) para VPH-2. Sete (6,7%)
pacientes apresentaram mais de um tipo de VPH detectado. Os sintomas mais frequentes foram
tosse e taquipneia, semelhantes a outras infecções respiratórias virais. As manifestações clínicas
não diferiram de forma significativa entre as infecções por VPH-1, -2, -3 e -4. Os VPH-1, -3 e -4
estavam presentes na população estudada ao longo dos três anos de vigilância, e o VPH-3 foi o
tipo predominante identificado nos primeiros dois anos.
Conclusão: Os VPHs contribuem substancialmente para a DRA pediátrica no Brasil com quase
30% dessa contribuição atribuível ao VPH-4.
© 2017 Sociedade Brasileira de Pediatria. Publicado por Elsevier Editora Ltda. Este é um artigo
Open Access sob uma licença CC BY-NC-ND (http://creativecommons.org/licenses/by-nc-nd/4.
0/).

Introduction and other common respiratory viruses in nasopharyngeal

aspirates.
Viruses are the predominant cause of acute respiratory ill-
ness (ARI) worldwide, and are responsible for substantial Methods
morbidity and mortality in children between 1 and 5 years of
age. Human parainfluenza viruses (HPIVs) account for a sig- The research ethics committee of Institute Biomedical Sci-
nificant proportion of viral ARIs in children, representing the ence of the University of São Paulo approved the study. From
second most common cause of upper and lower respiratory March 2008 to August 2010, nasopharyngeal aspirate sam-
tract infections, just after human respiratory syncytial virus ples were collected from patients under 2 years of age with
(HRSV).1 The four HPIV serotypes, HPIV-1, -2, -3 and -4, and ARI, attended to or hospitalized at the Santa Casa de Mis-
two subtypes, -4a and -4b, are estimated to cause up to 10% ericordia Hospital (São Paulo, Brazil), after written consent
of childhood ARIs.2 HPIV1 and HPIV2 are the primary cause was obtained from the children’s parents. The samples were
of croup in children aged 6---48 mons; HPIV3, and to a lesser placed in a viral transport tube and held up to 48 h at 4 ◦ C.
extent HPIV1, are more often associated with bronchioli- The samples were processed in a biosafety level 2 labora-
tis and pneumonia in children under 1 year. HPIVs also cause tory at the Institute of Biomedical Science, University of São
severe disease, including pneumonia and death in transplant Paulo. Total nucleic acids were automatically extracted from
recipients, as well as nosocomial infections and outbreaks, 300 ␮L of fresh specimen and eluted in 110 ␮L of RNase-free
similar to HRSV and influenza virus.3 elution buffer using NucliSENS easyMAG (bioMérieux---Brazil)
Little is known about the epidemiology and disease bur- according to the manufacturer’s instructions. Nucleic acids
den of HPIVs in the pediatric populations of Latin America, were kept frozen at −70 ◦ C until use. A multiple single-
especially in Brazil.1,4---6 There are even less studies con- plex rRT-PCR assay panel was used to detect and identify
cerning HPIV-4 infection in America since few laboratories HPIVs (types 1, 2, 3, and 4)8 and other human respiratory
provide specific diagnoses of HPIV-4. Because of its appar- viruses, including respiratory syncytial virus, human metap-
ently low prevalence and its difficulty of growth in cell neumovirus, adenovirus, and influenza viruses A and B.9---11
culture.7 The statistical analyses was conducted with Statgraphics
To better characterize the role of HPIV-4 and its clini- Centurion XV software; the chi-squared test for comparison
cal features in children under 2 years of age with ARI, real of proportions was used for each symptom to verify the pro-
time reverse transcription polymerase chain reaction (rRT- portion of patients presenting the symptom in the serotypes
PCR) analyses were used to identify four strains of HPIV analyzed. It also generated an analysis of means (ANOM)
556 Thomazelli LM et al.

plot to determine which samples were significantly different Discussion

from the grand mean. Since the p-value was greater than or
equal to 0.05, there are no significant differences between Despite associations between HPIV-4 infection and ARI hav-
the samples at the 95% or higher confidence level. ing been proposed for more than half a century,12 it is
not commonly included in molecular testing available in
Results Brazil because it is not considered to be a major disease-
causing virus.13 To the best of the authors’ knowledge, this
Nasopharyngeal specimens were collected from 1002 is the first report of HPIV-4 infection in children with ARI in
patients with ARI. Parainfluenza virus infection was labora- Brazil. This virus was circulating among pediatric patients
tory confirmed in 104 (10.4%) samples, of which 60 (57.7%) with ARIs during entire period tested, along with other
were positive for HPIV-3, 30 (28.8%) for HPIV-4, 12 (11.5%), respiratory viruses including other HPIVs. These results cor-
for HPIV-1, and two (1.9%) for HPIV-2. Most laboratory- roborate studies from other countries in which HPIV-4 was
confirmed HPIV-4 specimens were collected from August identified as the cause of respiratory disease in institution-
2008 to December 2008, but there were few cases col- alized children, as well as community acquired pneumonia
lected in 2009 and 2010, as shown in Fig. 1. The median age and bronchiolitis.14---16 HPIV-4 was the third most prevalent
of HPIV-4 infected patients was 7 months (ranging 0---24), virus (5.8%) in cases of adult influenza-like illness over two
8 months (1---24) for HPIV-3, 5 months (3---6) for HPIV-2, consecutive seasons in USA during 1998 to 2000.3 In this
and 11 months (6---22) for HPIV-1. In contrast, the HPIV-4 study, HPIV-4 prevalence lagged more common respiratory
frequency was higher in the age group of 3---6 months. Some- pathogens (RSV, adenovirus, influenza, and HMPV), but was
what lower to HPIV-3 and HPIV-1 detection, with greater the second most prevalent HPIV, following HPIV-3. Over-
number of cases between 7 and 12 months (Fig. 2). Medical all, HPIVs were the third most common respiratory viral
records of 87 HPIV infected patients were available and the pathogen detected.
clinical data are summarized in Table 1. Prominent clinical The analysis of age distribution according to viral infec-
symptoms included tachypnea (97.7%), cough (94.3%), dys- tion shows that the largest number of positive cases of all
pnea (94.3%), wheezing (87.4%), coryza (71.3%), and fever HPIVs, like other respiratory viruses, occurs in children aged
(67.8%). Thirteen (15%) of the infected patients required less than 1 year, which is consistent with the international
hospital admission (intensive care). In 23 cases, HPIV-4 was literature.8,9,17
the sole HPIV type detected. However, in the seven remain- When physical symptoms and clinical diagnosis were com-
ing cases, HPIV-1 (one case) and HPIV-3 (six cases) were also pared with etiology, no association could be found, so it was
detected. impossible to identify the HPIV type based only on clini-
Other respiratory viruses were detected in 473 (47.2%) of cal signs; however, a relatively higher rate of mechanical
the 1002 patient specimens. The most frequently detected ventilation and intensive care need was found in patients
viruses were RSV in 244 cases (24.3% of the total specimens), with HPIV-1 and 3, as well as a higher rate of cyanosis in
adenovirus in 121 cases (12.1%), influenza in 58 cases (5.8%), patients with HPIV-3. Among HPIVs, a higher rate of deaths
and human metapneumovirus (HMPV) in 50 cases (5%). Co- was observed in HPIV-1 cases (18.2%).
infections with two or more viruses were detected in 11.7% A limitation of this study was the end of sample collec-
of studied samples. tion before the end of last year studied, precisely in the

Seasonality of HPIVS
8

0
8

08

08

9
08

08

09

09

M 9

09

09

9
09

10

10

0
0

10
10
8

0
9
/0

l/0

/0

l/0
/0

/0

/0

/0

r/0

/0

/0

/0

/1

l/1
r/1
/0

/1
/0
g/

p/
b/

n/

g/
n/

b/

n/

g/

p/

n/

b/

n/
ar

ec
r

ct

ov

ar

ct

ov

ec

ar
ay

ay
ay
Ju

Ju

Ju
Ap
Ap

Ap
Fe

Ju

Au

Se

Fe
Ja

Ju

Au

Se

Ja

Fe

Ju

Au
O
O
M

M
N

N
D

D
M

Winter Spring Summer Winter Spring Summer

Fall Fall Fall
PIV1 PIV2 PIV3 PIV4

Figure 1 Seasonality of human parainfluenza viruses (HPIVs). Number of positive samples of each HPIV type by month of the year.
Human parainfluenza virus in pediatric patients 557

HPIV-1 HPIV-2

All age All age

19-24 19-24

13-18 13-18

7-12 7-12

03-6 03-6

0-2 0-2

2008 2009 2010 2008 2009 2010

HPIV-3 HPIV-4
All age All age

19-24 19-24

13-18 13-18

7-12 7-12

03-6 03-6

0-2 0-2

2008 2009 2010 2008 2009 2010

Figure 2 Frequency of each type of human parainfluenza virus (HPIV) detected by age groups (y-axis) and by time (x-axis). Graph
generated by the EPIPOI software.

Table 1 Clinical symptoms of HPIV positive patients (%).

Clinical symptoms HPIVs

HPIV-1(n = 11) HPIV-2(n = 2) HPIV-3(n = 50) HPIV-4(n = 24) All(n = 87) pa

Death 18.20 0.00 4.00 0.00 4.60 0.058
Cough 90.90 100.00 96.00 91.70 94.30 0.677
Paroxysm 9.10 0.00 6.10 0.00 4.60 0.396
Whooping 0.00 0.00 0.00 0.00 0.00 ---
Wheezing 81.80 100.00 88.00 87.50 87.40 0.855
Tachypnea 90.9b 100.00 100.00 100.00 97.70 0.033
Dyspnea 100.00 100.00 95.90 91.70 94.30 0.532
Cyanosis 9.10 0.00 20.00 8.30 14.90 0.352
Apnea/pause 0.00 0.00 4.00 8.30 4.60 0.524
Fever 90.00 100.00 60.00 78.30 67.80 0.077
≥39 ◦ C 40.00 0.00 16.70 13.00 17.20 0.140
Coryza 81.80 100.00 72.00 62.50 71.30 0.480
Hyaline 72.70 100.00 63.30 54.20 62.10 0.552
Purulent 18.20 0.00 10.40 8.30 10.30 0.674
Conjunctivitis 18.20 0.00 12.50 4.20 10.30 0.397
Diarrhea 9.10 0.00 12.00 0.00 8.00 0.212
O2 supply 72.70 100.00 84.00 79.20 81.60 0.657
Intensive care 18.20 0.00 18.00 8.30 14.90 0.532
Mec. vent. 18.20 0.00 16.00 8.30 13.80 0.616

There were six co-detections of HPIV-3 and -4, and one co-detection of HPIV-1 and -4.
n, number of positive patients with clinical information available.
a Level of significance of the chi-squared test for comparison of proportions (HPIV-2 was not included in the analysis).
b Presented a significant difference of the mean.

higher expected incidence period, affecting possible infer- There are no data describing HPIV-4 circulation in Brazil.
ences on the seasonality of HPIVs. Moreover, there was a lack Evidence was found of a moderate frequency of HPIV-4
of asymptomatic controls to help determine the prevalence detection (28.8% of HPIVs; 3% of all virus positive samples)
of subclinical HPIV4 infections, and only one hospital was in young children with ARI, equal to or higher than other
sampled. The restricted population complicates generaliza- HPIVs. Further studies are needed confirm these findings.
tion of data to the broader community. Longitudinal studies HPIV-4 should be included with other respiratory pathogens
should be performed to confirm the results obtained here. routinely tested for in developing countries, especially in the
558 Thomazelli LM et al.

group of high-risk infants, such as prematurity, congenital 4. Thomazelli LM, Vieira S, Leal AL, Oliveira DB, Souza TS, Golono
heart diseases, chronic lung disease, immune deficien- MA, et al. Surveillance of eight respiratory viruses in clinical
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Araújo-Neto CA, Oliveira JR, et al. The role of respiratory
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The findings and conclusions in this report are those of the
7. Canchola J, Vargosko AJ, Kim HW, Parrott RH, Christmas E, Jef-
author(s) and do not necessarily represent the official posi- fries B, et al. Antigenic variation among newly isolated strains
tion of the Centers for Disease Control and Prevention. of parainfluenza type 4 virus. Am J Hyg. 1964;79:357---64.
8. Morgan OW, Chittaganpitch M, Clague B, Chantra S, Sanas-
Funding sutipum W, Prapasiri P, et al. Hospitalization due to human
parainfluenza virus-associated lower respiratory tract illness in
rural Thailand. Influenza Other Respir Viruses. 2013;7:280---5.
CNPq, FAPESP. 9. Fry AM, Chittaganpitch M, Baggett HC, Peret TC, Dare RK,
Sawatwong P, et al. The burden of hospitalized lower respira-
Conflicts of interest tory tract infection due to respiratory syncytial virus in rural
Thailand. PLoS ONE. 2010;5:e15098.
10. Kodani M, Yang G, Conklin LM, Travis TC, Whitney CG, Anderson
The authors declare no conflicts of interest.
LJ, et al. Application of TaqMan low-density arrays for simul-
taneous detection of multiple respiratory pathogens. J Clin
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11. Heim A, Ebnet C, Harste G, Pring-Åkerblom P. Rapid and quan-
The authors thank the FAPESP, and the CNPq titative detection of human adenovirus DNA by real-time PCR.
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(Y0204004040131) for their financial support. They are
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grateful to Wladimir Jimenez Alonso and the NIH team for
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