Exhaled breath condensate sampling is not a new method for detection of respiratory viruses
Exhaled breath condensate sampling is not a new method for detection of respiratory viruses
Lieselot Houspie 0
Sarah De Coster 0
Els Keyaerts 0
Phouthalack Narongsack 1
Rikka De Roy 2
Ive Talboom 2
Maura Sisk 2
Piet Maes 0
Jannick Verbeeck 0
Marc Van Ranst 0
0 Laboratory of Clinical Virology, Rega Institute for Medical Research, Catholic University of Leuven , Minderbroedersstraat 10, B3000 Leuven , Belgium
1 Medical Doctor , Goudsbloemstraat 4, B3000 Leuven , Belgium
2 Medical Center, Catholic University of Leuven , Naamsestraat 80, B3000 Leuven , Belgium
Background: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. Methods: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. Results: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. Conclusion: Although very promising, EBC collection using the RTube is not reliable for diagnosis of respiratory infections.
Human respiratory tract infections represent the most
commonly encountered infections worldwide. In the
majority of cases, the etiology of these infections
remains undetermined due to rapid convalescence after
infection. Respiratory tract infections in healthy adults
can be caused by a variety of pathogens and the
detection of these agents is currently based on their isolation
from nasal swabs (NS), bronchoalveolar lavages (BAL),
nasopharyngeal aspirates and sputum samples. The
acquisition of these specimens by semi-invasive and
invasive techniques is often unpleasant for the patient.
Therefore, exhaled breath condensate (EBC) analysis has
recently been explored as a new and non-invasive
method to monitor lung inflammation and pulmonary
disease such as chronic obstructive pulmonary disease
(COPD), asthma, cystic fibrosis, lung cancer etc. EBCs
mainly consist of water vapour but a small fraction
contains respiratory droplets derived from the airway lining
fluid [1,2]. This observation has created a growing
interest in the use of EBC as a new sampling method for the
screening of respiratory viruses infecting the upper
airways. At first, investigators suspected that turbulence of
the inhaled air was responsible for the aerosolisation of
the respiratory fluid. However, the effect of the
turbulent airflow is limited to the upper airways since the
turbulent airflow becomes laminar as it reaches the smaller
bronchial airways and alveoli. Recently, the bronchiole
fluid film burst model has been described . This
model suggests that aerosols are produced during
inhalation by the bursting of fluid bubbles present in the
The aim of this study was to investigate whether the
EBC collection method was suited for the efficient
condensation of aerosolised virus particles during normal
breathing and to explore the isolation of respiratory
viruses in the condensate. Therefore we screened the
EBC samples with virus specific PCR assays targeting 14
respiratory viruses (Human Respiratory Syncytial Virus
(HRSV) A & B, Influenzavirus (Inf) A & B, Coronavirus
(CoV) NL63, E229 and OC43, Adenovirus (AdV),
Human Metapneumovirus (HMPV), Rhinovirus (RV)
and Parainfluenza virus (PIV) 1-4).
In this study, 102 EBCs were collected from otherwise
healthy volunteers showing respiratory or flu-like
symptoms (defined in Table 1), using a commercially
available condenser (RTube, Respiratory Research Inc.,
Charlottesville, Virginia, USA). The patient was
instructed to breath orally at tidal volumes into a
mouthpiece attached to a condenser for 10 minutes. No
nose clips were used during collection and saliva
contamination was avoided by the presence of a one-way
valve and the T-shaped section of the mouthpiece.
In a first part of the study that started during the
winter and spring of 2008/2009, 70 EBC samples were
collected from patients who voluntary presented
themselves to our laboratory. The majority of these
volunteers were students that responded to the
information leaflet, distributed in the university buildings of the
Catholic University of Leuven. The samples were
collected with the aluminium cooler sleeve chilled at -80C.
In the fall and first half of the winter of 2009/2010, 32
condensates were collected from patients who presented
themselves to their general practitioner. Due to practical
circumstances, the condensates were collected with the
cooler chilled at -20C. For 13 out of 32 collections, the
RTube was connected by a custom made
connectingpiece to the ECoVent (Jaeger, Germany). This device
registers ventilatory parameters such as the exhaled
volume, breathing frequency and tidal volume.
Additionally, a NS was obtained in parallel with the condensate
collection from each patient.
Table 1 Clinical manifestations of subjects
Figures represent the number of patients experiencing these clinical
manifestations. Figures between brackets are the percentages calculated to
total of 102 subjects.
All EBCs were immediately stored at -20C. Nasal
swabs (NS) were refrigerated. After viral DNA and RNA
extraction, EBC samples and nasal swabs were stored at
-80C. Three specimens were excluded from the study
due to incorrect condensate collection. A short
questionnaire was used to document the date of birth, the
severity of respiratory complaints and to record the days
of symptomatic illness from all volunteers. This study
was approved by the Medical Ethics Committee of the
University Hospital of Leuven and informed consents
were received from all participants.
Viral RNA and DNA extraction
Viral DNA and RNA were isolated with the QIAamp
MinElute Virus kit (Qiagen, Westburg, The Netherlands)
according to the instruction manual. EBC extracts were
eluted in 60 l elution buffer and NS extracts in 110 l
Virus detection and sequencing
The breath condensates were screened for 11 respiratory
RNA viruses (CoV NL63, E229 and OC43, RV, HMPV,
InfA&B and PIV1-4) [4-7] using a OneStep RT-PCR Kit
(Qiagen, Westburg, The Netherlands) in a 50 l reaction
containing 10 l of the extracted RNA, 0.6 M of
forward and reverse primers (Table 2), 1.5 l One Step
Enzyme Mix, 10 l 5 One Step RT-PCR Buffer and
400 M of each dNTP. For adenovirus screening, a
DNA PCR was carried out for which the amplification
reaction mix contained 0.5 M forward primer (AdFW)
and reverse primer (AdRV), 0.4 mM dNTPs, 10 l
Buffer C and 1 U Taq polymerase in a final volume of
50 l. The PCR primers used were located in conserved
regions of the genomes of the respiratory pathogens
(Table 2). The reactions were carried out in a T3000
Thermocycler 48 (Westburg, Leusden, The Netherlands)
with an initial reverse transcription step for RNA viruses
at 50C for 30 min, followed by PCR activation at 95C
for 30 s, 45 cycles of amplification followed by a final
extension step for 10 min at 72C. The DNA
amplification program was initiated with a denaturation step at
94C for 3 min, followed by 45 cycles of 94C for 30 s,
55C for 30 s and a final extension step at 72C for
1 min. The amplicons were subjected to a 6%
polyacrylamide gel and visualised under UV light by staining with
ethidium bromide. PCR products were purified using the
Invitek MSB Spin PCRapace Kit and cycle sequenced in
forward and reverse direction using the ABI PRISM
BigDye Termination Cycle Sequencing Ready Reaction kit
(Applied Biosystems, Foster City, CA, USA). Sequence
analysis was performed with the ABI3130 Genetic
Analyser (Applied Biosystems, Foster City, CA, USA).
Consensus sequences were obtained using the SeqMan II
software (DNASTAR, Madison, Wis.). For samples from
Table 2 Primers for viral screening
Sequence (5 3)
*Nucleotide positions for reference strain CoV NL63 (DQ445912).
patient 71 and 80, rhinovirus sequences were poor, but
sufficient to verify the virus by BLAST search. In addition,
the VP4/VP2 partial region was amplified to confirm
rhinovirus infection (data not shown). The nucleotide
sequences have been deposited to GenBank under
accession numbers [HM74039 to HM747056].
HRSV was detected using a RT-PCR assay as previously
described [8,9]. In brief, a multiplex mix was prepared in a
final volume of 25 l using 5 l extracted RNA, 12.5 l of
Eurogentec One-Step Reverse Transcriptase qPCR Master
Mix containing ROX as a passive reference, 0.125 l
Euroscript + RT & RNase inhibitor (Eurogentec, Seraing,
Belgium) 200 nM of HRSV-A and -B specific forward and
reverse primers and 100 nM of HRSV-A and -B MGB
probes. cRNA standards were constructed using the
MEGAshortscript T7 kit (Ambion, Austin, TX, USA) and
The viral load of RV positive samples were quantified
by qRT-PCR as described in the manuscript published by
Lu and coworkers . The Eurogentec One-Step
Reverse Transcriptase qPCR kit was used for preparation
of the master mix as described above. The primerset
and probe, located in 5UTR, were added to a final
concentration of 1 M and 0.1 M, respectively. cRNA
standards were constructed based on the PCR product of
sample 1 using the MegaScript kit (Ambion, Austin, TX,
USA). Quantification was performed with a
spectrophotometer at 260 nm and converted to the molecule number
. Tenfold serial dilutions, allowing detection in a
range of 8.6 106 to 8.6 102 RNA copies were used.
The RT-PCR assays were carried out on a ABI PRISM
7500 Sequence Detection System (Applied Biosystems,
Foster City, CA, USA). An initial reverse transcription
step was performed at 48C for 30 min, followed by a
denaturation step at 95C for 10 min. Finally, an
amplification step of 45 cycli at 95C for 15 sec and 1 min at
60C was completed.
A total of 102 EBCs were collected from volunteers
showing a symptomatic respiratory infection. Seventy
were collected during the winter and spring of 2008/
2009 and 32 during the fall and first part of the winter
of 2009/2010. The first group of participants consisted
of 42 (60%) women and 28 (40%) men and a median
age of 32 years (range 19 - 83 years) was observed. The
second group existed of 18 (56.3%) women and 12
(37.5%) men, with a median age of 29 (range 9 - 46
years). Age and gender was missing for 2 participants of
the second group. In total, 52% of the participants were
between 20-30 years old. Only 6% were younger than 20
years old and 3% were older than 70 years. In totality,
80 patients (78.4%) were already feeling ill for 1 to 7
days at the day the sample was obtained. Seven
volunteers (6.8%) were symptomatic for 8 to 14 days and 9
participants (8.8%) were already ill for more than 14
days at the day of sample collection. Data on the
duration of symptoms was lacking for 6 patients. Almost all
volunteers experienced at least 2 symptoms except for
two patients (Table 1). Forty-seven (46.1%) volunteers
complained about a constant runny or stuffy nose, 43
(42.2%) had frequent sneezing events and 38 (37.3%)
participants had a serious sore throat (Table 1).
In a first part of the study, we collected 70 EBCs.
Screening of the EBCs for 14 respiratory viruses (Table 2),
showed 5 RV (7.1%) positive samples (Table 3). In a
second part, we collected 32 EBCs from patients that
presented themselves to their general practitioner. Two
of these EBCs were positive for one of the 14 investigated
respiratory viruses, 1 for RV and 1 for InfB. To inspect
the detection rate of respiratory viruses in the
condensate, a NS was taken from this second group of
volunteers for comparison. In 15 out of 32 NS (46.8%), one or
more viral pathogens were isolated. Viral screening of the
Table 3 Viral screening of EBCs and NS
Days of illness at
time of collection
RV = Rhinovirus, RSV-B = Respiratory Syncytial Virus B, InfA = Influenza A, InfB =
Influenza B, N = Negative, - = No sample available, ND = viral load could not be
SD = standard deviation * single detection.
NS resulted in the detection of RV, InfA (subtype H1N1)
and HRSV-B. Quantification of the HRSV-B viral load
demonstrated for samples 72 and 101 viral titers of 8.0
104 RNA copies/ml and 6.8 107 RNA copies/ml
respectively. The RV RT-PCR assay did not allow the
quantification of all samples that tested positive for RV by PCR
(Table 3). Presence of the same pathogen in both the
EBC and the NS was confirmed for only 1 sample: sample
71, which tested positive for RV in both the EBC and the
NS. For sample 81, RV was detected in the NS and
analysis of the EBC demonstrated an InfB infection.
Viral generation rate
For EBC samples that were collected in the fall and
winter of 2009/2010, measurements with the ECoVent in
combination with the RTube were performed for 13
of the 32 collections. A mean volume of 763.5 litre air
(range 103.8 - 999.9) was exhaled during 5 min and
volunteers breathed with a mean breathing frequency of
20.8 (range 18.2 - 30.7) times per minute. Only 1 EBC
(Table 3, sample 81) was positive for InfB when using
the RTube in combination with the EcoVent. In
theory, the viral generation rate (number of viral RNA
copies exhaled per minute) can be predicted by
quantification of the exhaled viral load. Then, an estimation of
the RNA copies per litre exhaled air or per minute can
be calculated. Quantification of the exhaled InfB would
allow us to predict the generation rate for this virus.
Due to insufficient sample volume, we could not
determine the number of RNA copies in the sample.
Collection of exhaled breath condensates is a novel and
non-invasive method for obtaining samples of the upper
respiratory tract. The collection of EBC is easy to
perform and can be conducted in a home environment.
This method is much more agreeable for the patient
when compared to the unpleasant and invasive
collection of nasal swabs, BAL, aspirates, etc. This aspect
renders the method very attractive for routine laboratory
diagnostics of viral infections. Most studies that perform
breath analyses for viral detection use modified face
masks, with a removable central region in electret or a
removable Teflon filter on which exhaled particles
impact [12-14]. With the RTube collection device,
aerosolized particles of the airway lining fluid are
precipitated into a condensate when the breath is cooled
which serves as an immediate starting point for
Until now, this is the study with the largest subset of
volunteers that investigated EBC as a specimen for the
detection of respiratory viruses. Previous studies
reported the inclusion of a limited subset of participants
and investigated the presence of a limited number of
viruses in the breath samples. The study performed by
Fabian and colleagues, included 12 volunteers .
Huynh and co-workers recruited 9 volunteers for
exhaled breath sampling . In the study by
StelzerBraid et al., 50 EBCs were analysed  and St-George
et al. report the participation of 12 adults . These
studies have focused on the detection of InfA and -B,
PIV1-3, HRSV and HMPV, while we have screened the
samples for a panel of 14 commonly circulating
respiratory viruses. Based on the analysis of 99 EBCs (3 EBCs
were excluded), our results support the exhalation of
RV and InfB in 7% of our samples. Since many of the
volunteers had already been experiencing symptoms for
1 to 7 days, we initially presumed that they were already
recovering from the infection and were no longer
exhaling the virus. For common cold infections it is
suggested that a person may already be infectious for 1 or 2
days before experiencing any symptoms. However, in a
second part of our study we started collecting EBCs in
parallel with nasal swabs from patients presenting
themselves to their medical doctor, 1 to 3 days after onset of
symptoms. Only for 1 condensate the same pathogen
was detected in both the EBC and the NS. The
detection rate for respiratory viral pathogens in the NS was
46.8% which is much higher than the 7% detection rate
in the EBCs. The low detection of virus positive
condensates can therefore not be attributed to the fact that
volunteers were no longer infectious. The discrepant
detection rate between samples may also be explained
by different severity of respiratory infection, since
comparator samples were of different parts of the respiratory
tract. Patients that delivered a positive NS may have
possibly suffered from an upper airway infection
whereas EBC positive volunteers may have experienced
a more advanced, lower respiratory tract infection.
However, the effect of nasal inhalation on EBC collection,
guiding formed particles in the upper respiratory tract
to the lower compartments, in stead of oral inhalation
was not investigated. Patients with positive EBC samples
were experiencing symptoms for maximum two days at
the time of collection. However, this was not different
for 7 patients with positive NS. Six patients that
provided positive NS were experiencing symptoms for a
longer period at the time of collection (Table 3). In the
group of volunteers that provided an EBC negative or
EBC and NS negative sample, the manifestation of
symptoms were reported ranging from 1 day to more
than two weeks. When reported symptoms were
compared between EBC positive patients (7) and NS positive
patients (15), 27% and 33% in the positive NS group
experienced shivering and muscle pain whereas this
symptom was not indicated by any patient of the EBC
positive group. In all groups fever, headache, watering
eyes, stuffed nose, frequent sneezing, sore throat and
coughing were reported.
Volunteers were not diagnosed with other pathogens
before participation in the study. Since we did not test
these samples for other than viral pathogens, we can not
exclude the possibility that some of the negative NS are
positive for bacteria or other pathogens causing
respiratory illness. Recently, one study reported a detection
rate of 5% for influenza in EBC . This is in the same
range of the detection rate that we report for respiratory
viruses in general. Other studies with a limited number
of patients, describe a markedly higher sensitivity of 33
to 36% [12-14] but the higher percentage may be due to
the low number of participants subjects were included
. Remarkably, the studies reporting this higher
detection rate used collections masks, while the study
using the RTube reported comparable findings. Face
masks consist of electret which trap viruses based on
permanently charged fibres . In addition, the Teflon
filter has 2 m pores which will retain all larger
particles. Possibly, the lower detection rate can partly be
explained by the fact that the RTube is manufactured
in polypropylene and does not possess a virus attracting
and filtering feature like the aforementioned materials.
The qRT-PCR developed by Lu and coworkers for the
detection of RV, did not allow the assessment of the viral
load present in the EBC samples . Also for 4 NS, the
viral titer remained undetermined, probably due to the
limited sensitivity of the assay. For diagnosis, more
sensitive methods might be necessary to detect respiratory
viruses present in EBC since it is unpredictable how
diluted the viral particles in the specimen are. Recently,
nested qRT-PCR assays have been developed to allow a
more sensitive detection of viruses in aerosols .
Also person-dependent factors, such as the number of
particles produced, the exhaled volume and the age of
the patient, have been suggested to play an important
role for exhalation of viral particles. The participants
that were recruited in the study of Fabian and
coworkers were 12 years of age and older . For
hospitalized children a much higher rate of virus positive
samples is reported . In our study, the majority of
volunteers were between 20 and 30 years old. Only two
children less than 10 years and 3 elderly people (> 70
years) were included. One of the children tested positive
for InfA in the NS, but the infection was not confirmed
in the EBC.
For influenza, an exhaled generation rate of <3.2 to 20
influenza RNA copies per minute was predicted by
quantifying the virus aerosols that impacted on a
removable Teflon filter of a collection mask . We used
the RTube in combination with the ECoVent, that
allowed the registration of additional ventilation
parameters such as breathing frequency and exhaled volume.
In this way, when the number of RNA copies in the
EBC is quantified, the amount of viral particles that are
exhaled per litre or per minute can be estimated.
Unfortunately, we were not able to predict a virus generation
rate for InfB since viral load remained undetermined.
Although an inventive, new and promising method,
EBC collected by the RTube does not appear to be
appropriate for diagnosis of respiratory infections.
Nonetheless, this method may provide an alternative for
current sample procurement for epidemiological studies of
circulating viruses. This technique also confirms the
observation that viruses are able to disseminate through
normal breathing, particularly RV.
In addition, EBC collection from patients during
respiratory infections may be further investigated for
biomarker patterns. In calves that were experimentally
infected with bovine RSV, an increase in leukotriene B4,
indicating oxidative stress, was observed. This increased
level was also associated with the development of
bronchial hyperresponsiveness . In humans, a transiently
elevated H2O2 level was observed during common cold
infection. This marker returned to baseline values when
volunteers recovered from infection. H2O2 has also been
recognized as an interesting marker in asthma, where it
is associated with chronic lower airway inflammation
. In InfA infected volunteers, an increased CO level
was observed during upper respiratory infection. This
observation might imply that CO is an indicator of
airway inflammation or represents one of the host defence
mechanisms against viral infection . Therefore, a
better identification of the biomarker signature in
condensates of individuals experiencing a viral infection
might imply interesting findings towards the
identification of markers reflecting inflammation or antiviral
protection. This may contribute to the biomarker profiles
established for diseases like asthma and COPD, for
which viral infections are suggested to trigger or
exacerbate symptoms .
List of Abbreviations
AdV: Adenovirus; BAL: Brochoalveolaire lavage; COPD: Chronic Obstructive
Pulmonary Disease; CoV: Coronvirus; EBC: Exhaled Breath Condensate; HMPV:
Human Metapneumovirus; HRSV: Human Respiratory Syncytial Virus; InfA:
Influenza A; InfB: Influenza B; NS: Nasal Swab; PIV: Parainfluenzavirus; RV:
Rhinovirus; qRT-PCR: quantitative Reverse Transcriptase Polymerase Chain
We would like to thank the volunteers for their cooperation and interest in
our study. This research was supported in part by a grant of the Institute for
the Promotion of Innovation by Science and Technology in Flanders (IWT) as
part of SIMID, project number 060081, and by the European Community
under the EPIWORK grant (EC-ICT contract 231807). Piet Maes is supported
by a postdoctoral grant from the Fonds voor Wetenschappelijk Onderzoek
(FWO)-Vlaanderen. We thank our colleagues of the Laboratory of Clinical
Virology for helpful comments and critical reading of the manuscript.
LH designed, executed and coordinated the study. SDC contributed in the
sample acquirement and laboratory analysis. EK, PM and JV participated in
the execution of the study and added helpful suggestions during
preparation of the manuscript. PN cooperated in this study and participated
in the recruitment of volunteers and sample collections in a clinical setting.
RDR, IT, MS work as medical doctors at the Medical Center of the University
Leuven and supported our study by recruiting volunteers for our study. All
author have read and approved the manuscript.
The authors declare that they have no competing interests.
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