Human rhinovirus infection in young African children with acute wheezing
BMC Infectious Diseases
Human rhinovirus infection in young African children with acute wheezing
Heidi E Smuts 0
Lesley J Workman 2
Heather J Zar 1
0 Division Medical Virology/NHLS, Department of Clinical Laboratory Sciences, University of Cape Town , Cape Town , South Africa
1 Department of Paediatrics and Child Health, University of Cape Town, Red Cross War Memorial Children's Hospital , Cape Town , South Africa
2 Division of Clinical Pharmacology, Department of Medicine, University of Cape Town , Cape Town , South Africa
Background: Infections caused by human rhinoviruses (HRVs) are important triggers of wheezing in young children. Wheezy illness has increasingly been recognised as an important cause of morbidity in African children, but there is little information on the contribution of HRV to this. The aim of this study was to determine the role of HRV as a cause of acute wheezing in South African children. Methods: Two hundred and twenty children presenting consecutively at a tertiary children's hospital with a wheezing illness from May 2004 to November 2005 were prospectively enrolled. A nasal swab was taken and reverse transcription PCR used to screen the samples for HRV. The presence of human metapneumovirus, human bocavirus and human coronavirus-NL63 was assessed in all samples using PCR-based assays. A general shell vial culture using a pool of monoclonal antibodies was used to detect other common respiratory viruses on 26% of samples. Phylogenetic analysis to determine circulating HRV species was performed on a portion of HRV-positive samples. Categorical characteristics were analysed using Fisher's Exact test. Results: HRV was detected in 128 (58.2%) of children, most (72%) of whom were under 2 years of age. Presenting symptoms between the HRV-positive and negative groups were similar. Most illness was managed with ambulatory therapy, but 45 (35%) were hospitalized for treatment and 3 (2%) were admitted to intensive care. There were no in-hospital deaths. All 3 species of HRV were detected with HRV-C being the most common (52%) followed by HRV-A (37%) and HRV-B (11%). Infection with other respiratory viruses occurred in 20/128 (16%) of HRV-positive children and in 26/92 (28%) of HRV-negative samples. Conclusion: HRV may be the commonest viral infection in young South African children with acute wheezing. Infection is associated with mild or moderate clinical disease.
Wheezing is a frequent manifestation of lower
respiratory tract infection (LRTI) in infants and young
children. Viral infections are the commonest cause of acute
wheezing. Several respiratory viruses, including
respiratory syncytial virus (RSV), influenza viruses,
parainfluenza viruses, enteroviruses, human coronaviruses,
human metapneumovirus and human bocavirus have
been associated with wheezy illness [1-5].
With the improvement of molecular techniques the
frequency of HRV detection in clinical samples has
increased dramatically [6-8] providing increasing
evidence that HRV infection may be associated with LRTI
including bronchiolitis, pneumonia, asthma
exacerbations or influenza-like illnesses . A recent
populationbased study showed that HRV was detected in 26% of
children under 5 years of age hospitalized with
respiratory symptoms or fever . Subsequently, studies in
high income countries have confirmed the importance of
HRV as a cause of severe LRTI in young children
requiring hospitalization [11,12]. It is unclear if the diverse
spectrum of clinical illnesses associated with HRV
infection is related to host factors, the infecting HRV type
or both. Recent evidence suggests that infection with
HRV-C may result in more severe disease [12-16].
Since the first isolation of HRV in 1953 
approximately 100 serotypes have been described and new types
are being discovered indicating that this genus is
considerably more varied than previously recognized. Based on
sequence analysis, antiviral susceptibilities and receptor
usage HRV was until recently divided into 2 groups;
HRV-A and HRV-B . However, a third and possible
fourth grouping, HRV-C and HRV-D, have been
identified after sequence analysis of HRV types identified some
which did not cluster with HRV-A, HRV-B or other
species within the genus Enterovirus [15,16,19-23]. HRV-C
has a global distribution, with a prevalence intermediate
with HRV-A and HRV-B .
The importance of HRV as a cause of acute wheezing
illness in infants and young children has not been
studied in African children. The aim of this study was to
investigate the prevalence of HRV in African children
with acute wheezing.
A prospective study of children aged 2 months to 5 years
presenting with acute wheezing to Red Cross War
Memorial Childrens Hospital (RCCH) from May 2004 to
November 2005 (2 winter seasons) was undertaken.
RCCH is a public paediatric tertiary hospital in Cape
Town, South Africa that provides care to children mostly
from poor socio-economic backgrounds. Children were
eligible if they had a history of cough or difficulty
breathing within the prior 5 days and expiratory wheezing on
auscultation or hyperinflation of the chest. Exclusion
criteria were known underlying cardiac or chronic
pulmonary disease (other than asthma), presence of stridor
or daily treatment with oral corticosteroids for more than
2 days prior. Eligible children were sequentially enrolled
from Monday to Friday during working hours. Clinical
and sociodemographic information were recorded.
Written, informed consent was obtained from a parent
or guardian. The study was approved by the Human
Research Ethics Committee of the Faculty of Health
Sciences, University of Cape Town, South Africa.
A nasal swab was obtained using a dry sterile cotton
swab inserted sequentially into each nostril to a depth
of 2-3 cm and slowly withdrawn in a rotating motion as
recommended by the WHO guidelines on the collection
of human specimens . The tip of the swab was
placed in viral transport medium and transported to the
Virology laboratory on the same day. After a
clarification step (2000 rpm for 7 minutes) the medium was
stored at -20C.
RNA was extracted from 200 l of the respiratory sample
using the Talent Seek Viral RNA kit (Talent Sri, Trieste,
Italy) according to the manufacturers instructions. The
purified RNA sample was converted into cDNA using
random primers (Roche Diagnostics GmbH, Mannheim,
Germany) and the iScript cDNA synthesis kit (Bio-Rad,
CA, USA). Samples were screened for HRV using PCR
primers targeting the 5 untranslated region (5UTR)
which had previously been shown to amplify all known
serotypes and HRV-C species . In the first reaction
10 l cDNA was added to a 50 l PCR mix containing
2 IU Supertherm polymerase (JMR Holdings, Kent, UK),
15 mM Tris-HCl (pH 8), 50 mM KCl, 1.5 mM MgCl2,
200 mol/L each dNTP (Roche Diagnostics GmbH,
Mannheim, Germany) and 0.2 mol/L P1-1 and P3-1 primers
. Amplification was performed on a Thermo Hybaid
PxE 0.2 thermal cycler (Thermo Scientific, Waltham,
MA, USA), with the following conditions: 1 cycle of 94C
for 2 minutes, 40 cycles of 94 C for 15 s, 50C for 25 s,
and 72C for 35 s, and a final elongation step at 72C for
7 minutes. The second round semi-nested PCR was
performed on 2.5 L outer PCR product using the same
basic master mix ingredients containing 0.2 mol/L P1-1,
P2-1, P2-2, and P2-3 primers . Cycling conditions
were as for the first round PCR with an increase in
annealing temperature to 55C. Amplified products were
separated by electrophoresis in 2% agarose gel, and
visualized under UV irradiation after staining with ethidium
bromide. The expected sizes of the outer and inner HRV
PCR products were 390 bp and 300 bp respectively. VP4/
VP2 amplification was performed as above on a selection
of HRV-C and HRV-A samples to differentiate HRV-Ca
and HRV-Cc variants using the primers described by
Huang et al. . All work was performed in an
ISO15189 accredited molecular laboratory which employs
strict precautions to prevent contamination.
Detection of other respiratory viruses
A general shell vial culture using a pool of monoclonal
antibodies detecting respiratory syncytial virus (RSV),
influenza A and B viruses, adenovirus and parainfluenza
viruses 1, 2 and 3 was performed on every 4th sample (n =
58) by an indirect immunofluorescence assay (Light
Diagnostics, Chemicon International, CA, USA). Further
specific virus identification on pool-positive samples was not
undertaken. All samples were screened for human
metapneumovirus (hMPV), human coronavirus-NL63
(HCoVNL63) and human bocavirus (HBoV) by RT-PCR and PCR
as previously described .
Sequencing and Phylogenetic analysis
The HRV 5UTR and VP4/VP2 PCR products were
purified with a QIAquick PCR purification kit (Qiagen, Hilden,
Germany) and sequenced on an ABI 310 sequencer with a
fluorescent dye terminator kit (Applied Biosystems, Foster
City, CA, USA). The nucleotide sequences were aligned
with known HRV sequences from GenBank, including
HRV Ca and Cc reference strains , using CLUSTALX
software. A neighbour-joining phylogenetic tree was
constructed using the Treecon software program (version
1.3b) with 500 bootstrap resamplings . GenBank
accession numbers are HM623207-HM623277.
Continuous variables were expressed as median and
inter quartile ranges and compared using Kruskal-Wallis
Test. Categorical characteristics were analysed using
Fishers Exact test. A p-value of < 0.05 was considered
Two hundred and twenty children, median (25th-75th
percentile) age 12.2 (6.2-27.5) months, were enrolled
and had a nasal swab taken. More than half, 114
(51.8%), were under 12 months, while 163 (74.1%) were
less than 24 months of age, Table 1. HRV was detected
in 128 (58.2%) of the samples. Most HRV-positive
children were under 24 months of age (92/128; 71.8%)
while half were under 12 months. There was no
difference in the previous number of wheezing episodes in
HRV-positive compared to negative children. The
clinical symptoms and HRV status of children admitted to
hospital and those treated as an out-patient is presented
in Table 2. Wheezing was more common in hospitalized
children (93%) and the total duration of symptoms was
HRV was detected throughout the year although there
was a peak in spring (September-November of 2004 and
2005) and another in February-April 2005 (autumn)
Co-infection with hMPV, HBoV and HCoV-NL63 was
found in 8 (6.3%), 6 (4.7%) and 1 (0.8%) of the 128
HRV-positive samples respectively. Of the 92
HRVnegative samples 5 (5.4%) were hMPV positive, 10
(10.9%) HBoV positive and 3 (3.3%) HCoV positive. Of
the 58 samples screened for the common respiratory
viruses by general shell vial culture 14 (24.1%) were
positive; 4 (6.9%) HRV-positive and 10 (17.2%)
Sequencing of the 5 UTR was performed on 71/128
(55.5%) positive samples using convenience sampling
ensuring each month of the study period was included,
but without prior knowledge of patient data. Figure 2
shows the phylogenetic distribution. HRV-C can be
divided into 2 variants, Ca or Cc, with either
HRV-Alike or HRV-C-like 5UTRs respectively. HRV-Ca
sequences (n = 24) were interspersed within the HRV-A
region of the phylogenetic tree. The remaining HRV-C
samples (n = 13) formed a separate branch with the
Table 1 Clinical and demographic details of HRV-positive and negative children
n = 128 (58.2%)
n = 92 (41.8%)
OR (95% CI)
Table 2 Clinical profile of children admitted to hospital compared to those treated as an out-patient
n = 71 (32.3%)
n = 149 (67.7%)
OR (95% CI)
HRV positive status n (%)
HRV-C-like 5UTR. HRV-C was the most prevalent (37/
71; 52.1%) followed by HRV-A (26/71; 36.6%) and
HRVB (8/71; 11.3%). HRV-C was found throughout the year
while HRV-A had peaks in the spring and autumn
seasons and HRV-B was sporadically detected (Figure 3).
Presenting symptoms or signs in HRV-infected and
uninfected children were similar, except for fever which
was less common in HRV-infected group of children
and vomiting which was more common (Table 1). Most
children with HRV infection had mild illness, although
45 (35.2%) required hospitalisation and 3 (2.3%) were
admitted to the intensive care unit. The median (IQR)
duration of hospitalisation was 1 (1-4) days; there was
no difference in the duration of hospitalisation between
HRV-infected and uninfected children. There were no
in-hospital deaths. Of those admitted to hospital and
where a HRV sequencing result was available (n = 15) 8
(25.8%) were typed as species C, 5 species A (16.1%)
and 2 species B (6.4%).
HRV was the commonest viral infection in young African
children presenting to a tertiary childrens hospital in
South Africa with acute wheezing. HRV has previously
been reported in older children with wheezing or asthma
exacerbations [5,29]. However, there is now accumulating
evidence that HRV-associated illness occurs in young
children with LRTI with many studies from other areas
of the world reporting HRV detection rates from 24% to
48% [11,30,31], and as high as 78% in predisposed
Figure 1 Monthly distribution of HRV-positive and negative children.
Cc species obtained from the GenBank database.
children . Consistent with these, 72% of the
HRVthird required hospitalization. The study suggests that
positive children were under 2 years. This data is also
HRV is responsible for a large burden of illness in young
consistent with prior studies that report mild to moderate
African children with associated implications for health
illness in most children , although in this study a
HRV not typed
Figure 3 Monthly distribution of the different HRV species.
HRV has also been associated with recurrent wheezing
and with the development of asthma in older children
[32-35]. HRV-induced wheezing during the first 3 years
of life was associated with a 10 fold risk of developing
asthma by six years in a USA cohort of infants at high
risk for developing asthma . Asthma is the most
common chronic childhood illness in South African
children affecting 10-20% of adolescents; the prevalence
has increased over the last decade . The role of
HRV in the inception and development of asthma and
in triggering acute asthma episodes in African children
needs further study.
Consistent with other studies, HRV was identified
throughout the year with peaks of activity in the spring
of 2004 and 2005 and autumn of 2005 [3,10,35].
Interestingly this pattern was more prominent with HRV-A
than HRV-C and HRV-B which were detected
throughout the year or sporadically. Seasonal variation is a
common feature of many respiratory viruses, including RSV,
influenza, parainfluenza virus, and although the causes
of this variation are largely unknown, one possible
explanation may be as a result of viral interference
where one respiratory virus predominates and prevents
other viruses from establishing infection at the same
time. As the study was of 18 months duration any
inferences about seasonality are difficult to make. However,
the study period included 2 winter seasons. Further long
term studies of the epidemiology of viral infections and
HRV specifically in African children are needed.
Phylogenetic analysis of the 5UTR of HRV-positive
samples showed the circulation of all 3 species in Cape
Town, South Africa. In this study, and as reported by
Huang et al. , HRV-C was the most prevalent
followed by HRV-A and HRV-B. This distribution pattern
is different from other studies where HRV-A was more
frequently detected; this may vary depending on the
population [21,24,37]. Consistent with previous studies
HRV-C was the common type in children with wheezing
and asthma [13,21,37]. Typing using the 5UTR may
underestimate the true number of HRV-C samples as
there is evidence that recombination with HRV-A
species at this site is a common occurrence, resulting in
these types grouping with HRV-A species instead of
HRV-C [19,38]. However, limited sequencing of the
VP4/VP2 region (data not shown) and inclusion of
sequences of HRV-C with HRV-A-like 5UTRs in the
phylogenetic analysis confirmed that HRV-C was the
most common species in this population, with HRV-Ca
more prevalent (65%) than HRV-Cc. Further
confirmation is that all HRV-Ca sequences from this study
grouped phylogenetically with GenBank-derived HRV
isolates that have been confirmed to be true HRV-C
types based on the classification proposed by Simmonds
et al . As most children presented with mild or
moderate illness, and as only a subgroup of HRV
isolates were sub-typed, we were unable to investigate the
association between HRV species and clinical severity;
this requires further investigation.
The study has some limitations. A control group of
asymptomatic or clinically well children from the same
time period were not included. However, previous
studies have reported that HRV is more frequently found
in sick children than in asymptomatic children [4,11].
HRV can be shed from the nasophaynx for 10 to 14
days in immunocompetent individuals with extended
shedding in immunocompromised patients . The
immune status of children enrolled in this study was
not recorded, although it can be inferred that some
children may have been HIV-positive as South Africa is
a high HIV burden country. Thus detection of HRV in
some children with wheezy illness may therefore be an
incidental finding due to extended shedding. Due to
resource limitations, only a subset of specimens could
be tested for the common respiratory viruses (RSV,
influenza virus A and B, parainfluenza viruses 1, 2, 3
and adenovirus) using the relatively insensitive general
shell vial culture assay which does not provide sufficient
information to determine the role of co-infection and
disease severity. Finally, the study did not test for all
known respiratory viruses (e.g. human coronavirus 229E
and OC43, and human parainfluenza type 4) and
recently discovered viruses (e.g. human coronavirus
HKU1 and polyomaviruses K1 and Wu) which may also
play a role in wheezy illness.
HRV may be the commonest virus in young African
children with acute wheezing. Infection is associated
with mild or moderate clinical disease. Longitudinal
studies with frequent samplings and HRV type
identification are needed to investigate the role of HRV in
wheezing illness in African children and their long term
We thank the research nurses Ms. M. Roux and Ms. A. Joachim and Dr S
Streun for enrolment of children and the Virology laboratory staff at C18
Groote Schuur Hospital for performing the indirect immunofluorescence
shell vial assays. We are grateful for the support of the staff in the
ambulatory section of Red Cross Childrens Hospital.
HS conceived the study, carried out experimental studies, analyzed the
sequence data and drafted manuscript. LW performed data management
and statistical analyses. HZ conceived the study, obtained funding,
supervised the clinical study and participated in manuscript preparation. All
authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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