Viruses and bacteria in acute asthma exacerbations--a GA² LEN-DARE systematic review.

Abstract

A major part of the burden of asthma is caused by acute exacerbations. Exacerbations have been strongly and consistently associated with respiratory infections. Respiratory viruses and bacteria are therefore possible treatment targets. To have a reasonable estimate of the burden of disease induced by such infectious agents on asthmatic patients, it is necessary to understand their nature and be able to identify them in clinical samples by employing accurate and sensitive methodologies. This systematic review summarizes current knowledge and developments in infection epidemiology of acute asthma in children and adults, describing the known impact for each individual agent and highlighting knowledge gaps. Among infectious agents, human rhinoviruses are the most prevalent in regard to asthma exacerbations. The newly identified type-C rhinoviruses may prove to be particularly relevant. Respiratory syncytial virus and metapneumovirus are important in infants, while influenza viruses seem to induce severe exacerbations mostly in adults. Other agents are relatively less or not clearly associated. Mycoplasma and Chlamydophila pneumoniae seem to be involved more with asthma persistence rather than with disease exacerbations. Recent data suggest that common bacteria may also be involved, but this should be confirmed. Although current information is considerable, improvements in detection methodologies, as well as the wide variation in respect to location, time and populations, underline the need for additional studies that should also take into account interacting factors.

Full text

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Braido, Fulvio; Genoa University, Allergy and Respiratory Diseases,
Dept of Internal Medicine
Brusselle, Guy; University of Ghent, Department of Respiratory
Diseases
Canonica, G. Walter; Genoa University, Dept of Internal Medicine







Viruses and bacteria in acute asthma exacerbations – A
GA2LEN-DARE* systematic review


Journal: Allergy
Manuscript ID: ALL-2010-00734.R1
Wiley - Manuscript type: Review
Date Submitted by the
Author:
12-Oct-2010
Complete List of Authors: Papadopoulos, Nikolaos; University of Athens, Allergy Dpt, 2nd
Pediatric Clinic
Christodoulou, Ioannis; University of Athens, Allergy Dpt, 2nd
Pediatric Clinic
Rohde, Gernot; Maastricht University Medical Center, Department
of Respiratory Medicine
Agache, Ioana; SC Theramed SRL, Allergy and Clinical Immunology
Almqvist, Catarina; Karolinska Institutet, Dept of Woman and Child
Health; Karolinska Institutet, Dept of Medical Epidemiology and
Biostatistics
Bruno, Andreina; Consiglio Nazionale delle Ricerche, Allergy Centre
Bonini, Sergio; Consiglio Nazionale delle Recerche, .
Bont, Louis; University Medical Center, Pediatric Infectious Diseases
and Immunology 7. Pediatric Infectious Diseases and Immunology
Bossios, Apostolos; University of Gothenburg, 8. Krefting Research
Centre, Sahlgrenska Academy
Bousquet, Jean; Hôpital Arnaud de Villeneuve, Centre Hospitalier
Universitaire
Keywords :
respiratory virus, atypical bacteria, detection method, PCR, asthma
exacerbation



Allergy

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30. Department of Dermatology and Allergy, Allergy Centre Charité, Berlin, Germany
1
Viruses and bacteria in acute asthma exacerbations – A GA2LEN-
DARE* systematic review

N.G. Papadopoulos1, I. Christodoulou1, G. Rohde2, I. Agache3, C. Almqvist4, A. Bruno5, S.
Bonini6, L. Bont7, A. Bossios8, J. Bousquet9, F. Braido10, G. Bruselle11, G.W. Canonica10, K. H.
Carlsen12, P. Chanez9, W.J. Fokkens13, M. Garcia-Garcia14, M. Gjomarkai5, T. Haahtela15, S. T.
Holgate16, S.L. Johnston17, G. Konstantinou1, M Kowalski18, A. Lewandowska-Polak18, K.
Lødrup-Carlsen12, M. Mäkelä15, I. Malkusova19, J. Mullol20, A. Nieto21, E. Østerberg-Eller22, C.
Ozdemir23, P. Panzner19, T. Popov24, S. Psarras25, I. Roumpedaki1, M. Rukhadze26, A. Stipic-
Markovic27, A. Todo Bom28, E. Toskala29, P. van Cauwenberge11, C. van Drunen13, J.B.
Watelet11, M. Xatzipsalti1, P. Xepapadaki1, T. Zuberbier30.

1. Allergy Department, 2nd Pediatric Clinic, National and Kapodistrian University, Athens, Greece
2. Maastricht University Medical Center+, Department of Respiratory Medicine, Maastricht, Netherlands
3. Transylvania University, Dept. of Allergy and Clinical Immunology, Brasov, Romania
4. Karolinska Institutet, Dept of Medical Epidemiology and Biostatistics, Stockholm Sweden
5. Istituto di Biomedicina, Consiglio Nazionale delle Ricerche, Palermo, Italy
6. Consiglio Nazionale delle Recerche, Rome, Italy
7. Pediatric Infectious Diseases and Immunology University Medical Center, Utrecht
8. Krefting Research Centre, Sahlgrenska Academy, University of Gothenburg, Sweden
9. University Hospital, Montpellier, France
10. University of Genoa, Allergy and Respiratory Department, Genoa, Italy
11. Ghent University Hospital,Department of Otorhinolaryngology, Gent, Belgium
12. Oslo University Hospital, Rikshospitalet,Norway
13. Academic Medical Centre, Department of Otorhinolarygology, Amsterdam, The Netherlands
14. Pediatrics Department, Severo Ochoa Hospital, Madrid, Spain
15. Department of Allergy, Skin and Allergy Hospital, Helsinki University Hospital, Finland
16. Respiratory Cell and Molecular Biology Research Division, University of Southampton, UK
17. Imperial College London, National Heart and Lung Institute, UK
18. Medical University of Łódź, Department of Immunology, Rheumatology and Allergy, Łódź, Poland
19. Medical Faculty of the Charles Universit, Dept. of Allergology and Immunology, Pilsen, Czech Republic
20. ENT Department, Hospital Clínic – IDIBAPS, Barcelona, Catalonia, Spain
21. Pediatric Allergy Unit, Children's Hospital La Fe, Valencia, Spain
22. Department of Dermatology, Odense University Hospital, Odense, Denmark
23. Marmara University Medical Faculty, Division of Pediatric Allergy and Immunology, Istanbul, Turkey
24. Medical University, Clinical Centre of Allergology of the Alexandrovska Hospital, Sofia, Bulgaria
25. Biomedical Research Foundation, Academy of Athens, Greece
26. Centre of Allergy and Immunology, Tbilisi, Georgia
27. Department of Clinical Immunology, Pulmonology and Rheumatology, Sveti Duh General Hospital,
Zagreb, Croatia
28. Coimbra University, Coimbra, Portugal
29. Finnish Institute of Occupational Health, Helsinki, Finland

Short title: Microbes associated with acute asthma

Keywords: Respiratory virus, atypical bacteria, detection method, PCR, asthma exacerbation
Abbreviations: AAE = Acute Asthma Exacerbation; CP = Chlamydophila pneumoniae; hBoV =
Human bocavirus; hCoV = Human coronavirus; hMPV = Human
metapneumovirus; LRI = Lower Respiratory tract Infection; MP = Mycoplasma
pneumoniae; PIV = Parainfluenza virus; PCR = Polymerase Chain Reaction;
RSV = Respiratory Syncytial Virus; RV = Rhinovirus; URI = Upper
Respiratory tract Infection

* GA2LEN: Global Asthma and Allergy European Network
DARE: Definition And Risk factors of Exacerbations working group

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Correspondence:
Nikolaos G. Papadopoulos
Allergy Research Centre,
2nd Pediatric Clinic,
University of Athens,
41,Fidippidou str.,
11527 Athens, Greece
ngp@allergy.gr
+30 210 7776964
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Abstract

A major part of the burden of asthma is due to acute exacerbations. Exacerbations have been
strongly and consistently associated with respiratory infections. Respiratory viruses and bacteria
are therefore possible treatment targets. In order to have a reasonable estimate of the burden of
disease induced by such infectious agents on asthmatic patients, it is necessary to understand
their nature and be able to identify them in clinical samples by employing accurate and sensitive
methodologies. This systematic review summarizes current knowledge and developments in
infection epidemiology of acute asthma in children and adults, describing the known impact for
each individual agent and highlighting knowledge gaps. Among infectious agents, human
rhinoviruses are the most prevalent in regard to asthma exacerbations. The newly identified
type-C rhinoviruses may prove to be particularly relevant. Respiratory syncytial virus and
metapneumovirus are important in infants, while influenza viruses seem to induce severe
exacerbations mostly in adults. Other agents are relatively less or not clearly associated.
Mycoplasma and Chlamydophila pneumoniae seem to be involved more with asthma persistence
rather than with disease exacerbations. Recent data suggest that common bacteria may also be
involved, but this should be confirmed. Although current information is considerable,
improvements in detection methodologies, as well as the wide variation in respect to location,
time and populations, underline the need for additional studies that should also take into account
interacting factors.
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parainfluenza virus, respiratory syncytial virus, rhinovirus, Chlamydophila or Mycoplasma) and
4


Introduction

In recent years, a growing number of observations have highlighted the importance of
respiratory infections in acute asthma exacerbations. Respiratory viruses have repeatedly and
consistently been associated with asthma exacerbations in different patient groups, with
detection frequencies ranging from 40% to 90%. Bacteria, mostly Mycoplasma and
Chlamydophila pneumoniae, are frequently identified in chronic asthma, and may also
precipitate exacerbations. Taking into account that a major proportion of asthma-related disease
burden is due to exacerbations [1], as well as that infectious agents are potential treatment
targets, it becomes evident that the above associations require further attention. In order to have
a reasonable estimate of the burden of disease induced by infectious agents on patients with
asthma, it is necessary to understand the nature of these agents and be able to identify them in
clinical samples by employing sensitive and accurate methodologies [2]. Newer detection
techniques are developing at a fast pace and will be the topic of a subsequent review. Newly
discovered infectious agents are filling some of the knowledge gaps in epidemiology.
Nevertheless, it is still uncertain whether the microbial epidemiology of asthma reflects viral
epidemiology in the community, or whether some of the agents, most notably human
rhinoviruses (RV), possess specific ‘asthmagenic’ properties [3]. The purpose of this review is
to summarize current knowledge and developments in microbial epidemiology of acute asthma
in children and adults, describing the known impact for each individual agent.
The strategy to generate the bibliography for this review was the following: the PubMed
online bibliographic database was queried for the combinations of keywords: 1) “x AND
wheezing”, 2) “x AND acute asthma” and 3) “x AND asthma exacerbation” (where “x” was
adenovirus, bocavirus,
coronavirus, enterovirus, influenza virus, metapneumovirus,
limited to: Humans, English, published in the last 10 years. The reference lists generated from
the above searches were merged into one database using Endnote X2 software (Thomson Corp.),
giving rise to a total of 654 and 121 unique articles for the viruses and the two bacterial species,
respectively. Subsequently, reviews, letters and editorials were removed and the remaining
original articles (456 and 85 respectively) were examined for relevance to microbial
epidemiology. Finally, a number of articles of historical importance from the 90’s, were added
in order to generate the final bibliography of this review.


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remarkable genetic and antigenic heterogeneity and comprise over 100 serotypes conveying
5
Association of viral infections with acute asthma
The first studies investigating the specific viral aetiology of acute expiratory wheezing in
children were already performed over 40 years ago. However due to the limited sensitivity of the
methods used, the frequency of viral detection was low, ranging from 14 to 49% [4]. With the
introduction of molecular detection methods, the role of viruses in acute asthma exacerbations
(AAE) has been increasingly appreciated. Typically, in a French study of children hospitalized
for a severe AAE, PCR detected a viral agent in about 82% of the cases, while more traditional
methods (virus culture and immunofluorescence) identified only 34% [5]. Using molecular
detection, between 62% and 95% of children with acute wheezing episodes test positive for
respiratory viruses (with most studies reporting a prevalence of over 80%), in both hospital and
community settings [6-16] [11, 17-20]. The prevalence in adults is less, but still in the range of
41 - 78% [21-25]. This may be due to real differences, or to the fact that adults produce and shed
less infective virus and/or that recent studies using PCR are not many.
RVs are the most frequently detected virus type at all ages, with the exception of infants
hospitalized with bronchiolitis, where respiratory syncytial virus (RSV) prevails. Most other
agents have small to moderate involvement. The relative contribution of specific agents is
described below. A summary of the data relating infectious agent prevalence and exacerbations
of acute asthma is provided in Tables 1 and 2. Although co-infections with more than one
infectious agent are not infrequent, and there are suggestions that multiple infections may
increase the risk of asthma [26], there is still not enough evidence to evaluate the role of such
occurrences.

Rhinoviruses
Human rhinoviruses are 7.2 – 7.5 kb, single-stranded, positive-sense, non-enveloped
RNA viruses. They belong to the Enterovirus genus within family Picornaviridae. RVs exhibit
serotype-specific immunity. They have been grouped on the basis of phylogenetic analysis of
viral protein VP4/2 coding region, into two groups, HRV-A and HRV-B. Recently, a novel
distinct genetic cluster, HRV-C, has been identified [27-30]. Although type-C variants have not
yet been cultured in vitro and therefore cannot be formally assigned into distinctive serotypes,
existing genetic sequence data suggest that 16 – 28% of human RVs belong to this new clade
[28, 31].
RVs are the agents most frequently associated with the common cold as well as asthma
exacerbations, with proportions reaching up to 80% [32]. In children, most studies have included
a wide age-range (3 - 6 months to 13 - 16 years) and samples are of nasal origin. RVs are always
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Furthermore, recent evidence suggests that early RV infection is strongly associated with
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dominant in emergency/hospital settings (28% in Sweden [12], 25% [10], or 52% (including
enteroviruses) [10] in Finland, 47% in France [5], 65% in Greece [33], 58% in Korea [13], 71%
in the USA [7], 82% in South Africa [34]). In the community, 46%-50% of wheezing episodes
in 9 - 11 year-old children during a one-year period could be attributed to RV [6, 35]. In studies
where a control group has been included in the design, RVs are most frequently the only
pathogen significantly higher in cases, possibly due to the small number of other pathogens,
whether compared with normal subjects where odds ratios range from 2 - 10 [7, 11, 19],
controlled asthmatics [18], or patients with upper respiratory tract infection (URI) [36].
Variability within the above studies can be explained by differences in methodologies, sampling,
seasonality, duration, age distribution and more. It should be pointed out that in temperate
climates RV induces disease mostly in the fall and spring, in association to return from school
holidays and is the major pathogen behind the September asthma epidemic [18]. Seasonality
may be different in tropical climates [37].
In recent years, the importance of RV in wheezing episodes in infants and its association
with subsequent asthma has been revisited. It appears that RV, rather than RSV, are the agents
more frequently associated with wheezing, even in infants. In a birth cohort from Australia
intensively monitoring for respiratory infections, 39% of all wheezing episodes in the first year
of life were associated with RV, three times as many as those associated with RSV [14]. A
similar proportion has been reported before [9]. In a noteworthy study, Malstrom and colleagues
used bronchial biopsies of 47 infants (4 – 26 months old) with recurrent asthma-like respiratory
symptoms to conduct in situ hybridisation for RV. RVs were detected in 45% of specimens.
Abnormal lung function (decreased airway conductance) was significantly more common
among RV-positive infants (86%) than in RV-negative ones (58%). Occurrence of a respiratory
infection in the six weeks preceding bronchoscopy significantly correlated with RV positivity
[38].
asthma symptoms and/or persistence later in childhood, once again more than RSV [9, 16, 17].
The duration of such associations awaits further follow up of these cohorts.
The time course of RV infection in AAE is still not completely understood. It seems that
RV may induce early viremia, probably in more severe cases [33], and may persist for several
weeks [34, 39], although this has been challenged with the possibility of frequent re-infections
[40].
In adults the pattern is similar, although the literature is less extensive. In South-African
adults with acute severe asthma who required hospital admission, RVs were detected in nasal
aspirates by PCR in 35% on admission, in 16% after 7 days, in one patient after 28 and 56 days
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More studies are needed in order to evaluate the contribution of enteroviruses in AAE.
7
and were absent in controls [41]. In a North-American study, 29% of asthma exacerbations in
adults were associated with RV in a longitudinal arm and 36% in a cross-sectional one [22].
Somehow lower prevalence has been reported from Australia (8% and 11% in hospitalized
patients or in the emergency room respectively) [24, 42]. A study from Singapore on adult
patients with near-fatal asthma requiring mechanical ventilation and patients with acute asthma,
found RVs (picornaviruses) in lower respiratory samples in 47% and 28%, respectively [23].
A considerable number of studies have recently focused on the role of the newly
identified RV-C group, and suggest that a major proportion of all RV infections are RV-C
infections [27, 43, 44] [45, 46]. Wheezing or AAE are common clinical manifestations of RV-C
infections [27] [44-46]. In 142 asthmatic children over 2 years of age, RV-C comprised one third
of RV-positive cases and was found exclusively in children with an AAE [31]. Interestingly, a
strong interfering effect of RV-C infections on susceptibility to co-infection with other RNA
respiratory viruses, especially RSV, has been reported [47].

Enteroviruses
Enteroviruses also belong to the family of Picornaviridae and share many of their viral
features with RVs [48]. Often the differentiation between enteroviruses and RVs is difficult, as
many studies use methods that detect picornaviruses without differentiating between RVs and
enteroviruses. Direct data are still lacking in adults. In cohort studies of exacerbating children
the reported prevalence is 9.8 - 18%, mostly evaluated by PCR [5, 8, 9, 39]. Higher prevalence
has been reported in one study which assessed 293 hospitalized children over two years (25%)
[10] and in another study that evaluated samples only during the summer (29%) [36].
In two case-control studies, prevalence was lower (< 5%) and not significantly different
from controls, although the studies were probably not powered adequately for such comparison
[11, 19].

Coronaviruses
Human coronaviruses (hCoV) are single-stranded positive-sense RNA viruses.
Historically two different groups were distinguished, represented by hCoV-229E and hCoV-
OC43 [48]. Recently, two more strains relevant to AAE, hCoV-NL63, [49] and hCoV-HKU1
were discovered [50]. Coronaviruses are regarded as important common cold agents. Overall,
reported respiratory infection rates for hCoV among children usually range from < 1% to 9%
with varied distribution between subtypes [13, 20, 51-55].
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in around 20 - 25% of cases [23, 24, 42, 61], while less have been reported in severe patients (6 -
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Early findings suggested an association of hCoV with AAE in 9 - 11 year-old children
(13%) [6] and adults (12 - 21%) [22]. However, more recent studies, all using PCR with or
without additional methods, have yielded rather low frequencies. In children, some studies were
unable to identify hCoV at all [9, 18], while others did so at a level of 1 - 4.5% [5, 10, 20].
Frequently, hCoV infections are co-infections with other viruses [20]. Similar numbers were
found for the newer strain hCoV-NL63 in pre-school children from South Africa (2.4%) and
Korea (1.3%) [13, 56]. In case-control studies, hCoV was not present more frequently in
wheezing children in comparison to controls [11, 14, 19]. In a study of 49 adults presenting to
the emergencies with AAE, hCoV was found in 4% of induced sputum samples [24].
Overall, hCoV seem to have a minor, if any, contribution to AAE.

Influenza viruses
Influenza viruses belong to the orthomyxoviruses. Influenza produces yearly epidemics
during the winter months; this should be taken into account in the interpretation of the studies on
AAE. For example, when sampling AAE during September, no influenza cases were found
(controls 1%) [18], while the highest reported proportion comes from infants during the flu
season (20%) [11]. Nevertheless, most studies in children report an overall low prevalence,
ranging from close to zero [13, 19], up to 7% in a one year cohort in the community [6], with a
median of around 4% [5, 8, 10, 20]. However, among children with asthma, exacerbations due to
influenza may lead to hospital admission, and healthcare utilization attributable to influenza is
high [57-59]. The 2009 pandemic H1N1 influenza virus has also been reported to induce severe
exacerbations in patients with asthma, mostly children [60], however, the detailed kinetics of
this epidemic in relation to asthma is outside the scope of this review.
In contrast, prevalence in adults with AAE appears to be higher. Several studies from
patients in the emergency department or hospitalized with AAE have identified influenza viruses
8%) [22, 23]. The differential response between pediatric and adult asthmatic patients to
influenza viruses is interesting, it needs however confirmation through case-control studies,
before it can be helpful in designing public health strategies in respect to influenza protection.

Parainfluenza viruses
Parainfluenza viruses (PIV) belong to the Paramyxoviridae family. In studies evaluating
viral involvement in AAE in children, PIV have been found in 0 - 7.4% of cases (median 4%)
[5, 6, 8, 10, 13, 19]. However, case-control designs have failed to show significantly increased
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with some studies finding very low prevalence (1.5 - 3.6%) [8, 19] and other at the range of 20 -
9
prevalence in comparison to either non-wheezy lower respiratory tract infection (LRI) or URI
[14, 15], healthy controls [11], or stable asthmatics [18].
In adults, while one longitudinal cohort found PIVs in 18% of AAE in the community, it
failed to identify any in patients at the emergency department [22]. Similarly, no PIV was found
in near-fatal asthma or AAE in Singapore [23]. In summary, PIVs are detected at low frequency
in AAE in children; an association is doubtful. No conclusion can be drawn yet in adults.

Respiratory syncytial virus
Respiratory syncytial virus (RSV) belongs to the genus Pneumovirus within the family
Paramyxoviridae. There are two known serotypes A and B. Most infections occur from
December to February and it is the main pathogen associated with severe bronchiolitis in infants
[48, 62, 63]. Prevalence of RSV in acute wheezing can be dramatically different depending on
age, but also between studies. RSV has been associated with at least four different wheezing
phenotypes: acute bronchiolitis, post-bronchiolitis, the inception of asthma and AAE [64]. In
infancy and early childhood differentiation between bronchiolitis, acute wheeze and AAE is
difficult, making studies with a wide age range equally difficult to interpret.
In infants, prevalence can vary from as low as 16% [9], to as high as 54 - 68% [7, 10,
20]. It should be noted however, that the majority of these infants were or could be diagnosed as
bronchiolitis. In a birth-cohort study from Australia, RSV was the second most frequently
detected virus type (after RV) in wheezy LRI cases (16.8%) [14]. Similar results in a community
setting have been reported from the UK (27%) [65].
In pre-school children hospitalized for wheezing, prevalence can remain as high as 30%
[66], but can be lower (7.4%) [13]. In older children RSV has been associated with 4 - 12% of
AAE [6] [18, 34].
In studies including a wide age range results are not consistent and difficult to interpret
40% [5, 10, 11].
In adults with acute asthma early studies have reported a low proportion (1.3 - 3%) [22,
42]. In contrast, two more recent studies from Australia found RSV in 37% and 39% of AAE in
the emergency department using induced sputum samples [24, 61]. However, in older (> 65
years) adults, where RSV is believed to reappear as an important infectious pathogen, RSV
accounted for 7.2% of hospitalizations for asthma [67], while no RSV could be detected in near-
fatal asthma in Singapore [23].
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RSV or influenza infection [73]. In South Africa, among the ‘novel’ respiratory viruses (hMPV,
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In summary, RSV is a major viral pathogen inducing wheezing episodes in infants and
preschool children with a frequency of up to 70%. RSV may also be important in older children
and adults, however, we cannot yet explain the high variability reported in the literature.

Human metapneumovirus
Human metapneumovirus (hMPV) is a recently discovered respiratory virus belonging,
like RSV, to the genus Pneumovirus within Paramyxoviridae [68]. Frequencies of degtection pf
hMPV in RTI varies from 2 - 3% to just over 20%, with most studies reporting 5 - 9% and virus
activity peaking in winter/early spring, similarly to the seasonal distribution of RSV. Symptoms
reported are also similar to RSV and include URI, bronchiolitis and pneumonia. In a large
Australian study conducted over four consecutive years, over 10,000 NPA specimens from
patients with acute lower RTI were tested. Of these, 7.1% were hMPV-positive, with 92% of
hMPV-positive children being < 5 years of age [69].
Infection with hMPV has been associated with AAE and wheezing, mostly in young
children. The majority of studies have been performed in hospitalized pediatric populations:
when inclusion was because of an asthma exacerbation, hMPV has been detected in around 4%-
8% [10, 13, 19, 62], however this was not necessarily higher than the control population [19].
Increased impact has been observed in younger children (1.9% versus 0% in infants younger
than three months versus those 3 - 12 months old [70] or 8.9% in children younger than three
years in comparison to 7.5% in the whole population of 133 children [71]. When study inclusion
was based on specific infection rather than clinical presentation, the proportion who wheezed
with hMPV was 25% in a French study and 55% in an Israeli one, significantly higher in
comparison to RSV or influenza, respectively [63, 72]. Slightly lower prevalence rates have
been reported in children presenting to emergency departments (hMPV positive in 2.2 - 2.8%)
[36, 73]. Exacerbations were more frequent in children with hMPV, in comparison to those with
hCoV-NL63 and hBoV), hMPV was found to be the most prevalent (8.3%), with a frequency
peaking for children aged 7 - 9 months old [56].
In a birth-cohort study from Australia, wheezing was present in 29% of the 329 episodes
of LRI within the first year. hMPV was detected in 3% of LRI in general, and was 10 times
more likely to be associated with non-wheezy LRI (n-wLRI), than wheezy (wLRI) [14]. In a
later report from the same group describing a subpopulation of the original report, hMPV was
identified in 4.7% of n-wLRI and in 1.4% of wLRI cases in children at high atopic risk [15].
In adults, in a large group of hospitalized patients in the USA (1,386 adults, mean age:
75 years) hMPV incidence was 8.5% over four consecutive winters, with wheezing being a
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controls or healthy subjects [79]. Taking into account the quite high proposition of positives in
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common clinical characteristic (80%) in infected patients [74]. In another North-American
study, Williams et al have ascribed an etiologic role of hMPV infection in triggering AAE, since
none of the 6.9% of patients hospitalised for AAE who were hMPV-positive (by PCR) on
admission, tested positive three months after discharge [75]. In a study of 88 Egyptian adults
presenting, among others, with AAE (67%) hMPV was significantly associated only with
pneumonia [76].
In summary, hMPV is detected mostly in young children with wheeze, at the age where
AAE is difficult to distinguish from acute bronchiolitis, with a frequency < 10%. Its association
with asthma later on in life is less clear.

Adenoviruses
Adenoviruses are non-enveloped, double-stranded DNA viruses. Forty seven human
adenovirus types are classified and subdivided into six subgenera A to F. Infection may be
productive, abortive, or latent (chronic). In latent infections as well as in transformed and/or
tumour cells, viral DNA is integrated into the host genome [48]. Virus-host DNA recombinants
are also found in productive infections. Adenoviruses bind to a receptor called CAR (coxsackie
and adenovirus receptor) [77].
In wheezing children, adenovirus detection rates are 1.3 - 5% [5, 10, 13, 20], with
slightly higher frequencies (7 - 8%) in general diagnoses of acute RTI [51, 78].
In a case-control study of 133 children hospitalised for wheezing, no significant
difference was observed in adenovirus frequencies compared to controls (PCR) [11]. In two
other case-control studies of children hospitalised for asthma exacerbations the investigators
failed to detect adenovirus at all using PCR [18, 19].
In contrast to the above, in a study from Turkey, adenovirus DNA was detected in 20/28
(71.4%) of children with acute asthma exacerbation, while it was 33%-37% in asymptomatic
the control groups, these results should be interpreted with caution.
In adults, the frequency is low ranging from 0.7 - 2.5% of asthma exacerbations [22, 42].
In contrast in a study from Singapore on adult patients with near-fatal asthma requiring
mechanical ventilation and patients with acute asthma, adenoviruses were found in lower
respiratory samples in 24% and 3%, respectively [23]. These results suggest a minor role of
adenoviruses in AAE in adults with the exception of near-fatal asthma, where a special role
should be further investigated.


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Molecular diagnosis (PCR, RT-PCR) can also be used, however these assays are not yet
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Human bocavirus
Human bocavirus (hBoV) belongs to the Parvoviridae which are non-enveloped, single-
stranded DNA viruses [80]. Several studies in children, have used PCR to identify hBoV in
respiratory tract infections world-wide, with reported detection rates between 1.5% and 8%
(19% in one case) [12, 51, 54, 81, 82]. Co-infection with other viruses is fairly frequent (14-
60%), while in the small number of case-control studies the detection rate in healthy individuals
is almost zero.
In a study of 231 Korean children (1 month - 5 years) hospitalized with acute wheezing
hBoV was the third most frequently detected virus, after RV and RSV, with an incidence of
5.6% [13]. Similarly, Allander and colleagues identified hBoV as the sole viral agent in 5% of
259 children (3 months – 15 years; median age 1.6 years) hospitalized for acute expiratory
wheezing; mixed hBoV infection with other viruses was detected in 19% [12]. In 710 Spanish
children hospitalized with respiratory infection, hBoV was found in 13.9% (mostly infants), with
one third of these being single-positives. The virus was associated with recurrent wheezing in
half of the cases [83]. A similar frequency in acute wheezing (7.4%) was reported from South
Africa [56]. Reports so far indicate that hBoV may be among the agents associated with AAE,
but more studies are needed. It has been suggested that serological analysis and serum PCR, in
addition to nasopharyngeal material PCR, may be needed in order to optimize detection rates
[84].

Bacteria
Atypical bacteria such as Chlamydophila pneumoniae (CP) and Mycoplasma
pneumoniae (MP) represent an important cause of human respiratory tract disease, involved in
URI, acute bronchitis, exacerbations of chronic bronchitis, and pneumonia. Clinical diagnosis is
based on the micro-immunofluorescence test for CP, and specific IgM/IgG EIA for MP.
optimized, and cannot readily differentiate between acute and latent (chronic) infection.
A longitudinal study of 108 children (9 - 11 years old) with asthma symptoms, showed
no difference in the frequency of CP detection when sampling symptomatic (23%) or
asymptomatic periods (28%) [85]. Nevertheless, CP-specific secretory-IgA was much higher in
subjects with four or more exacerbations, compared to those with one, suggesting that chronic
CP infection is associated with the frequency of AAE. In a French study of 132 nasal aspirates
from 75 children hospitalized for a severe attack of asthma, CP and MP were identified by PCR
in 4.5% and 2.2%, respectively [5]. In another population of children hospitalized for severe
asthma MP was identified in 20% of 119 children with known asthma and in 50% of 51 children
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In contrast, a number of epidemiological studies of pediatric AAE report low or no
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hospitalized due to their first asthma attack. The frequency of CP was lower (3.4% and 6%,
respectively). Asthma recurred among 62% of those in whom an atypical pathogen was
diagnosed, but in only 27% of patients not infected with an atypical pathogen, suggesting an
effect of MP in initiation and recurrence of asthma [86]. Lehtinen et al determined bacterial co-
infections in 220 children hospitalized for acute wheezing with defined viral etiology. Bacterial
co-infections were more frequent (22%) for patients positive for RV, compared to children
positive for other respiratory viruses; MP was the second most frequent bacterial species
identified, with an incidence of 5% [87].
In a recent case-control study from Japan, 103 infants (3 - 23 months), hospitalized for
an acute episode of wheezing, and 64 healthy matched controls were tested for the presence of
CP by serology and reevaluated after one year. Wheezing infants had significantly higher CP-
IgM levels and were more frequently classified as CP-infected (44.6%), compared to controls
(17%), while asthma developed significantly more frequently in wheezing infants with than
without CP infection (70% vs 20.7%, respectively) [88]. In a case-control study from Italy, 71
children (2 - 14 years) presenting to the paediatric emergency department with an acute episode
of wheezing, had a significantly higher incidence of acute infection with MP (22.5%) and CP
(15.5%), than healthy controls [89]. During a three-month follow-up, recurrent wheezing was
significantly more frequent in non-antibiotic-treated children with acute MP and/or CP
infection, than in children without. In another study of 82 acute asthmatic children in northern
France, 5% were infected with CP and an additional 5% with MP. Persistent symptoms and
reduced recovery after three weeks were more frequently associated with atypical bacterial
infections compared to viral infections [8]. In a study from Turkey including 79 asthmatic
children (37 with acute asthma attacks, 42 with stable asthma) and 36 healthy controls,
significantly higher MP and CP specific-IgM was found between the acute asthma and the other
two groups [90].
detection of atypical bacteria. An Italian, six-month study of 85 infants hospitalized for a first
episode of wheezing detected MP as a single pathogen in only one child; CP was not detected at
all [20]. Low infections rates (≤ 4%) and no significant difference between controls and
asthmatic children during the September epidemic in Canada were reported by Johnston et al
[18]. A birth cohort study in Australia also reported low frequency of detection for CP and MP
(< 3%) with no significant difference between controls and patients [14, 15].
In adults, an early British study did not provide evidence for a significant association
between CP infection and acute asthma serologically comparing 123 patients hospitalized with
acute asthma versus 1,518 controls [91]. In contrast, CP infection was confirmed in 168
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