Molecular and Microscopic Analysis of Bacteria and Viruses in Exhaled Breath Collected Using a Simple Impaction and Condensing Method
et al. (2012) Molecular and Microscopic Analysis of Bacteria and Viruses in Exhaled Breath Collected Using a Simple
Impaction and Condensing Method. PLoS ONE 7(7): e41137. doi:10.1371/journal.pone.0041137
Molecular and Microscopic Analysis of Bacteria and Viruses in Exhaled Breath Collected Using a Simple Impaction and Condensing Method
Zhenqiang Xu 0
Fangxia Shen 0
Xiaoguang Li 0
Yan Wu 0
Qi Chen 0
Xu Jie 0
Maosheng Yao 0
Leo L. M. Poon, University of Hong Kong, Hong Kong
0 1 State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering, Peking University , Beijing , China , 2 Department of Infectious Disease, Peking University Third Hospital, Peking University , Beijing , China
Exhaled breath condensate (EBC) is increasingly being used as a non-invasive method for disease diagnosis and environmental exposure assessment. By using hydrophobic surface, ice, and droplet scavenging, a simple impaction and condensing based collection method is reported here. Human subjects were recruited to exhale toward the device for 1, 2, 3, and 4 min. The exhaled breath quickly formed into tiny droplets on the hydrophobic surface, which were subsequently scavenged into a 10 mL rolling deionized water droplet. The collected EBC was further analyzed using culturing, DNA stain, Scanning Electron Microscope (SEM), polymerase chain reaction (PCR) and colorimetry (VITEK 2) for bacteria and viruses. Experimental data revealed that bacteria and viruses in EBC can be rapidly collected using the method developed here, with an observed efficiency of 100 mL EBC within 1 min. Culturing, DNA stain, SEM, and qPCR methods all detected high bacterial concentrations up to 7000 CFU/m3 in exhaled breath, including both viable and dead cells of various types. Sphingomonas paucimobilis and Kocuria variants were found dominant in EBC samples using VITEK 2 system. SEM images revealed that most bacteria in exhaled breath are detected in the size range of 0.5-1.0 mm, which is able to enable them to remain airborne for a longer time, thus presenting a risk for airborne transmission of potential diseases. Using qPCR, influenza A H3N2 viruses were also detected in one EBC sample. Different from other devices restricted solely to condensation, the developed method can be easily achieved both by impaction and condensation in a laboratory and could impact current practice of EBC collection. Nonetheless, the reported work is a proof-of-concept demonstration, and its performance in non-invasive disease diagnosis such as bacterimia and virus infections needs to be further validated including effects of its influencing matrix.
Funding: This study was supported by the National Science Foundation of China (Grants 21077005 and 20877004), and National High Technology Research and
Development Program of China (Grant 2008AA062503). The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Bioaerosols are present virtually anywhere in the environment,
and their exposure is shown to cause numerous adverse health
effects . In addition, there is also a possible release of
biowarfare agents in a man-made bio-terror event. A number of
studies demonstrated that the respiratory tract can be colonized
with disease organisms . Through talking, coughing, sneezing
or singing, the potential virulent organisms can be exhaled and
spread into the ambient environment , which accordingly
causes air contamination. For example, SARS in 2003 and H1N1
in 2009 outbreaks were shown to be attributed to the airborne
route of disease transmission .
Among many other diseases, respiratory infection accounts for
23.342.1% of the total hospital infections , and is listed as the
third leading killer . However, present diagnosis procedures
using nasal swabs, bronchoalveolar lavages, nasopharyngeal
aspirates or sputum samples, appear to cause unpleasant
experiences in addition to long detection time. During flu
outbreaks, body temperature or isolation procedures are often
used to control and prevent further spread, however such methods
are lacking scientific evidence and not always effective with those
patients infected but in latent period. On another front, exhaled
breath condensate (EBC) as a simple and noninvasive method is
increasingly being utilized in early disease screening and infectious
aerosols measurements, e.g., lung cancer [13,14], asthma [15,16],
and other respiratory problems [17,18]. In previous studies,
human influenza A viruses were detected in exhaled breath using
EBC [19,20] as well as filter , mask [22,23] and a liquid
sampler . In another study, foot-and-mouth disease viruses
were also found in the exhaled air from experimentally infected
cattle . In addition, high levels of bacterial concentrations in
EBC were also observed in other studies . It was recently
shown that exhaled breath could be also analyzed for fungal
infection by relevant biomarker, e.g., 2-Pentyl furan (2PF) for
aspergillosis . Overall, EBC has demonstrated great potential
and advantages in early disease screening and diagnosis ,
opening a new arena for studying airway inflammation and
chemistry . Recently, Vereb et al (2011) suggested that exhaled
Figure 1. Exhaled breath condensate collection device (A) and method developed in this study: B) the EBC collection device cover,
C) the collection device base with a layer of ice and hydrophobic film on the top, D) the exhaled breath condensate collection
method: 510 ml DI water pipetted and scrolled over the hydrophobic film to scavenge EBC droplets.
breath can be also used for assessing a variety of environmental
For EBC related studies, the first key step is the collection of
exhaled breath. Over the years, a variety of devices (Table S1,
Supporting Information) were developed including Rtube
collection system (Respiratory Research, Inc, Charlottesville, VA) and
EcoScreenH condenser (Erich Jaeger Gmbh, Wurzbur, Germany).
Typically, these devices would be able to collect 1000 ml of EBC
samples within about 10 min, however the collection often comes
with a lengthy procedure and a higher cost. For example, use of
the EcoScreen involves 7 steps: 1) turn on to cool, 2) clean
collection tube, 3) clean condensation chamber insert, 4) retrieve
cooling sleeve from freezer, 5) sample collection, 6) sample storage
and transport, 7) removal of sample (Respiratory Research, Inc,
Charlottesville, VA). The RTube eliminates the first 3 steps, but
each collection still requires 10 min and costs $23.25 (Respiratory
Research, Inc, Charlottesville, VA) compared to 31 min and
$47.17 per collection for the EcoScreen. These collection devices
are generally expensive, e.g., the EcoScreen costs around $9000. A
recent study compared the sampling efficiency of the Rtube
(widely used EBC collection device) with that of throat swab
method, showing detection rates of 7% and 46.8% for the Rtube
and the throat swab method, respectively . It was suggested
that the RTube is not applicable for viral detection in exhaled
breath . In addition, condenser coatings , sampling
temperature  and sampling times  were shown to affect
physical collection efficiencies of available EBC collectors. Among
others, the noted problems with these available EBC collection
devices are the device availability, reusability and possible cross
contamination , which would negatively impact their wide
applications. In addition, EBC collection is strictly limited to the
method condensation only in most studies . To fully utilize
EBC in early disease screening, diagnosis and environmental
exposure assessment, simple yet efficient EBC collection device
using different methods and biological characterization of the EBC
sample are needed.
In this study, a novel EBC collection method was developed by
using hydrophobic surface, a layer of ice, and a droplet scavenging
procedure. The physical collection efficiency (amount of EBC
collected per unit of time) of the device was evaluated. In addition,
biological analysis and characterization of EBC samples collected
from human subjects were conducted using culturing, DNA stain,
SEM, qPCR and species identification tool VITEK 2. This work
contributes to the effort in applying EBC together with molecular
tools as a non-invasive method in rapid disease diagnosis.
Materials and Methods
Development of exhaled breath condensate collection
device and method
The collection method and device developed and experiential
set up for collecting EBC are shown in Figure 1 and Figure 2,
respectively. As observed in Figure 1, a simple EBC collection
device was developed here. The EBC collection device is
composed of four major parts as shown in Figure 1: collection
device cover, collection device base, a layer of ice, and a
hydrophobic film (treated by ultralow temperature 270uC). The
collection device cover and base were made of TeflonTM
polytetrafluoroethylene (PTFE) material, and a parafilm (Parafilm
Co. Menasha, WI) used as the hydrophobic surface. The
dimensions of the collection device are measured as 80640640
(mm) (length6width6height). In the collection device cover, there
is a hole with a diameter of 6 mm as the exhaled breath inlet. The
thickness of the collection device cover and base was about 3 mm,
and the whole collection device weighs around 105 g. The layer of
ice is used to keep the treated hydrophobic film cool.
For EBC collection, sterile water (DNA and RNA free) was first
added into the collection base of the device up to a depth of 5 mm
as observed in Figure 2. And then, the device base together with
the cover was placed in an ultralow temperature (270uC)
refrigerator (Thermo Fisher Scientific Co. Marietta, OH) to form
a layer of ice. Following this step, a sterile hydrophobic parafilm
measured as 8064060.3 (mm)(length6width6thickness) was
placed onto the surface of the ice suited in the collection base.
To collect EBC samples, a disposable sterile straw with a diameter
of 5 mm (16 cm long) is inserted through the exhaled breath inlet
shown in Figure 2, with its end 2 mm above the hydrophobic
parafilm. The human subjects are then advised to mouth-breathe
without wearing a nose clip through the exhaled breath inlet
shown in Figure 2 toward the hydrophobic film for a selected time
(14 min). Due to the low temperature and hydrophobic nature of
the parafilm surface, exhaled breath quickly condenses into tiny
liquid droplets on the hydrophobic surface. Assuming an average
breathing rate of 12 L/min for an adult, the particle speed from
the exhaled breath would be around 10 m/s given the size of the
straw (5 mm in diameter). Therefore, during the exhaled breath
collection, the bacteria or virus particles would impact onto the
hydrophobic surface at a speed of 10 m/s. In addition to
condensing used for other EBC collection procedures, the method
developed here also rely on the impaction to collect the bacterial
and viral particles. Given such a speed, there might be possible
particle bounce problems, however the bacterial or viral particles
in the exhaled breath usually come with water droplets, which thus
minimizes the potential particle bounce problem.
After the collection, about 10 ml of DNA and RNA free DI
water was pipetted onto the hydrophobic film as observed in
Figure 1. To collect breath samples on the hydrophobic film, one
only needs to use the pipette to touch the DI water droplet, and
then drag the DI water droplet to scroll over the entire surface.
The DI water droplet would move with the pipette without an
extra step. The materials collected on the surface would be
subsequently scavenged into the water droplet. After this
operation, the collected EBC samples in the form of bigger liquid
droplet as shown in Figure 1D were transferred to a sterile tube by
a pipette for subsequent analysis. The samples collected without
the exhaled breath from human subjects are used as the negative
controls. The EBC collection efficiency and biological analysis of
collected samples were performed as outlined in the experimental
procedure shown in Figure S1 (Supporting Information).
Amount and variability of EBC collected by the device
To investigate the amount of variability in EBC collected by the
method developed, six student volunteers were recruited to exhale
through the device for 1, 2, 3 and 4 min. The volume of collected
EBC was measured by a calibrated pipette (Eppendorf,
Hauppauge, NY). The amount of EBC per unit time collected by the
device was determined using averages of EBC samples obtained by
the volunteers under each of specific collection time tested. For
each EBC collection, a different hydrophobic film and a different
exhalation straw were used. In addition, the particle size
distributions in the exhaled breath through mouth-breathing were
also measured in a particle free bio-safety hood using an Optical
Particle Counter (OPC) (GRIMM Co. Ltd., Ainring, Germany) at
a flow rate of 1.2 L/min. To ensure air stream balance, the OPC
was connected to a two-way tubing, which connects to clean air
(Biological SafetyHood) and the breathing straw, respectively.
Bacterial and viral aerosol concentrations and species in
In this work, seven patients with onset flu symptoms (their
medical information is listed in Table S2, Supporting Information)
were also recruited from the respiratory clinic of Peking University
Third Hospital in Beijing. About 40 ml of exhaled breath
condensate collected from each of 7 patients was diluted by 10
times and then plated on Trypticase Soy Agar (TSA) (Becton,
Dickson and Company, Sparks, MD) plates at 26uC for 23 days,
and colony forming units (CFUs) were manually counted. The
total culturable bacterial aerosol concentration was calculated as
CFU/m3 (exhaled breath) by considering the collection time and
an average breathing rate of 12 L/min for an adult. Besides, the
culturable bacterial species were identified using VITEKH 2
(BioMerieux, Inc,100 Rodolphe Street, Durham, NC). In
addition, molecular detection of bacteria and virus using qPCR and
RT-qPCR, respectively, were performed according to the
procedures described in Supporting Information S1. To further
confirm the bacterial presence DNA stain of EBC sample by
Acridine Orange (AO) was also conducted.
The differences in collected EBC volumes and culturable
bacterial aerosol concentrations obtained by the EBC collection
device were analyzed by Analysis of Variance (ANOVA). A
pvalue of less than 0.05 indicates a statistically significant difference
at a confidence level of 95%. Collection of EBC from human
subjects was approved by Peking University Ethnics Committee.
Results and Discussion
Here, a novel EBC collection method and device was developed
and evaluated in collecting EBC samples from human subjects
using culturing and molecular methods. Compared to those
currently available devices shown in Table S1, our device is
lightweight with simplicity, reusability, and lower cost. The
developed collection device itself costs less than $10, with about
$0.5 for consumables (straw and hydrophobic film) per collection.
The time needed for 100 ml EBC including sample collection and
removal was around 2 min. The physical collection efficiency of
the device is shown in Figure 3. The data points shown in the
figure were averages of the EBC samples collected from six
volunteers under each of the collection times (1, 2, 3 and 4 min)
tested. In general, the amount of EBC sample collected was
observed to increase with increasing collection time were observed
among subjects. As also observed in Figure 3, the method has a
good reproducibility (small variations). ANOVA analysis indicated
that the collection time had a statistically significant effect on the
amount of EBC sample collected per unit of time
(p-valFigure 6. Determination of total bacterial aerosols in EBC by qPCR; DNA standards (STD) used were 3.15, 3.156101, 3.156102,
3.156103 ng/ml Bacillus subtilis DNA; Sample 17 represent EBC samples collected from seven human subjects with their medical
conditions listed in Table S2; DI water was used as the negative control.
ue = 0.0026). For the 4 min collection, the volume of collected
EBC (168.7 mL) was 1.8 times of that (60.0 mL in average) by
1 min. In our study, when no EBC was collected about 1 mL of
liquid was obtained from the hydrophobic surface in an
environment with a temperature of 17.919.3uC and a relative
humidity level of 4652%. In addition, during the breath sample
collection, the collection device had a higher air pressure due to
the exhaling, thus it is less likely that environmental air would
come into the device. This suggests that environmental water
vapor had limited impact on the collection method given the total
amount of EBC collected. A recent study indicated that the
minimum required volume of EBC was 50 mL for follow-up
biological and chemical analysis, such as multiplexed cytokine
analysis . This on the other hand implies that the EBC device
developed in this study can provide adequate amount of EBC
sample for rapid analysis. Here, only one type of hydrophobic
surface (parafilm) was tested, and in the future different
Figure 7. Dissociation curve of bacterial aerosols in EBC samples amplified by qPCR; Samples 17 were those collected from seven
human subjects with their medical conditions listed in Table S2; Bacillus subtilis species was used as the positive control and DI
water was used as the negative control; the curves shown here include two duplicates for each EBC sample.
Figure 8. SEM images (different resolutions) of bacteria in EBC samples and images of colony forming units after culturing; the EBC
samples were collected from human subjects and cultured using liquid broth overnight; different colored arrows point to likely
different bacteria (different morphologies); Sphingomonas paucimobilis, Kocuria rosea, Bacillus lentus, Aerococcus viridians, Bacillus
firmus, Kocuria kristinae, Staph. Xylosus were identified in EBC samples from patients with respiratory symptoms using VITEK 2 system.
hydrophobic materials should be also explored to improve the
As listed in Table S1, currently available EBC collection
devices, e.g., the Rtube and the EcoScreen, are comparable to
ours with respect to rate of EBC collection. However, our EBC
device has advantages in size, weight, and simplicity. In our study,
we used a 16 cm long straw for exhaling toward to the super
hydrophobic surface without any control of saliva for the possible
contamination. However, our collection time was only 14 min,
and during such short sampling period the sample contamination
by saliva is very limited given the length of the straw. Another
advantage of our developed device is the one time use of the
hydrophobic parafilm (disposable) and exhalation straw with an
easy collection of EBC, which thus prevents the possible cross
contamination and facilitates the collection of EBC samples from a
large number of subjects. This is particularly useful during an
influenza outbreak or a man-made bio-terrorism attack in which a
rapid screening of exposed persons needs to be conducted
Here, the EBC samples collected by the developed device from
seven human subjects recruited from a respiratory unit of Peking
University Third Hospital in Beijing were studied using culturing,
DNA stain, SEM and molecular methods. In this study, the
particle size distributions trends in a typical exhaled breath were
also measured and are shown in Figure 4. As observed in the
figure, the number concentration decreased with increasing
particle diameter. For bacterial size ranges (0.652.2 mm), a
concentration level of 329 to 25819 particles/L was observed,
while for larger particles of 2.24 mm a concentration level of 60 to
400 particles/L was obtained. In previous studies, similar particle
size distribution trend in exhaled breath was also found using the
OPC, although the droplet concentrations for respective size
ranges were slightly different [21,39]. Nonetheless, due to its rapid
evaporation water droplet itself or those adsorbing on bacterial
particles in the exhale breath will certainly affect the results
obtained here. The results from OPC indicated that particles of
larger than 2.5 mm only accounted for 0.4% of the total particles
exhaled. According to ICRP (1994), the total lung deposition
efficiency for particles larger than 2 mm is more than 80%, while
for smaller particles of less than 1 mm, the deposition efficiency is
less than 40%, i.e., 60% exhaled out . In addition, larger
particles could stick to the straw wall. Therefore, in the exhaled
breath as well as those collected into DI water droplet smaller
particles would dominate.
Figure 5 shows the concentrations of culturable bacterial
aerosols in EBC samples collected from seven human subjects.
As shown in the figure, bacterial concentration levels ranged from
693 to 6,293 CFU/m3. ANOVA tests indicated that there were
statistically significant differences in culturable bacterial aerosol
concentrations for EBC samples collected from different subjects
(p-value = .0001). In a recent study, human occupants are also
identified as the significant contributors for indoor bacteria, i.e.,
the emission rate is about 37 million gene copies per person per
hour, and a distinct indoor air signature of bacteria was
demonstrated to be associated with human skin, hair, and nostrils
. During human breathing, the bacterial particles from
environmental air are continuously inhaled, some of which, i.e.,
smaller ones, can be exhaled out again by the lung and reside with
nostrils. Here, bacterial species Sphingomonas paucimobilis and
Kocuria rosea were detected using Vitek2 in six EBC samples as
shown in Table S2. Because of limitation of Vitek 2, certain
bacterial species were not identified in our study. Among the
subjects, subject #6 had substantially higher culturable bacterial
concentrations than other subjects. From his medical conditions
shown in Table S2, it was likely that his fever was caused by the
bacterial infections. In his EBC sample, we found Kocuria variants
which were thought to cause catheter-related bacteremia . For
other human subjects, the culturable bacterial aerosol
concentration levels ranged from 700 to 3000 CFU/m3 and Sphingomonas
paucimobilis, a non-fermenting Gram-negative bacillus, were
detected. In a previous study, S. paucimobilis was found to cause
nosocomia bacteremia outbreak . For negative control
samples, we did not observe the bacterial growth, indicating no
contamination during the EBC collection. Ideally, bacterial
particles in EBC should be collected using a suitable size-selective
sampling tool to investigate the bacterial counts for different size
range. However, such device is currently not available yet.
Compared to the environmental culturable bioaerosol
concentrations, those in EBC samples collected had relatively higher levels,
thus representing an important source of bioaerosols particularly
in a high human occupancy environment. In addition to viruses,
Rhodococcus equi, a bacterium causing pyogranulomatous
bronchopneumonia, were detected in the exhaled air from foals in a recent
study . When pathogenic bacteria are breathed out, they could
pose a serious public health threat.
Figure 6 shows the qPCR amplification plot from EBC samples
collected from seven human subjects in a respiratory clinic. As
observed from the figure, bacterial samples were successfully
amplified (Ct values were 1619), while the positive sample (B.
subtilis) had a Ct value of 15 and the negative control had a value
of 28. Based on the DNA standards used, the concentrations of
bacterial DNA in the EBC samples (Sample 17) were in the range
of 0.32 mg/mL3.15 mg/mL. Detection of the bacterial DNA in
EBC samples was also confirmed by the melting curve of qPCR
amplification as shown in Figure 7. As observed in the figure, most
EBC samples had a peak at 68uC, the same as that of the positive
control B. subtilis. For a few different peaks observed, they might be
the possible primer dimer (PD) from the PCR non-specific
amplification process. In addition to the qPCR amplification of
bacteria in EBC samples collected, DNA stain (AO method) was
also performed and the results are shown in Figure S2. As
observed in the figure, both viable (green) and dead (yellow) were
found in the EBC samples collected and the positive control B.
subtilis samples, while no cells were detected in the negative
control. SEM images with different resolutions and agar plate
culturing shown in Figure 8 also indicated that EBC samples
(cultured) had various types of bacteria based on their
morphologies and colony color. From SEM images, it can be estimated
that most bacteria are in the range of 0.51.0 mm. According to
total particle deposition curve developed by ICRP (1994) ,
more than 60% of bacterial particles of below 1 mm could be
exhaled out. These smaller bacterial particles could remain
airborne for a prolonged time period, thus playing an important
role in airborne transmission of potential diseases. Results shown
in Figures 5, 6, 7, and 8 indicate that high levels of bacterial
aerosols were detected in the EBC samples collected, and the
results on the other hand also implied that the developed device
was efficient in collecting bacterial particles in the exhaled breath.
These experimental data further confirm that exhaled breath is an
important source of bacterial aerosols in the built environments.
In this study, qPCR was also applied to detecting influenza A
H3N2 viruses in EBC samples collected by the device. As observed
in Figure S3, H3N2 viruses were detected in the EBC sample
collected from subject #3 with a Ct value of 28, while those for
subject #1, #2 were shown below the detection limits. In
addition, spiking viruses into the samples in general enhanced the
overall qPCR signal as observed in Figure S3. This on the other
hand suggests no inhibition or amplification occurred when
amplifying H3N2 viruses in EBC samples using qPCR. According
to information shown in Table S2, subject #3 had a fever, but no
other information was available at the time of the experiment. In a
previous study, it was indicated that use of the RTube for EBC
collection had a very low viral detection rate (7%) compared to
nasal swabs (46.8%) . Recently, a mask-like sampler was also
tested and proved to be useful in detecting viruses using PCR in
exhaled breath . It was indicated that airborne virus detection
is difficult due to their low concentration and the presence of a
wide range of inhibitors, thus optimized molecular biology should
be performed to enhance their detection . Although the
number of the subjects tested is limited here, the developed
method, i.e., EBC collection and qPCR application, was
demonstrated successful in detecting viruses from human exhaled
breath. This would offer a non-invasive method for diagnosis of
respiratory infections by using EBC. In the future, more patients
should be tested with the EBC collection device developed here for
Exhaled breath holds great promise for monitoring human
health and for the diagnosis of various lung and systemic diseases,
but analysis challenges remain due to the complex matrix of the
breath [46,47]. In this study, different from available devices
restricted solely to condensation a simple and low cost EBC
collection method using impaction and condensing was developed
here for collecting bacteria and virus particles. An important
advantage is the reusability of the collection device with a
disposable hydrophobic film and an exhalation straw yet with a
rapid EBC collection. This would offer the opportunity to collect
EBC samples from a large number of subjects, especially during an
influenza outbreak or a man-made bioterrorism event, within a
shorter time frame. The developed EBC collection method was
shown highly successful in detecting bacteria in EBC samples in a
clinical setting. The developed EBC collection method was also
shown applicable in detecting influenza viruses too. Experimental
data here also suggest that exhaled breath, which was shown to
contain smaller bacterial particles, could play an important role in
airborne transmission of potential diseases. The collection
efficiency of other substances including bio-markers (NO,CO,
8isoprostane, hydrogen peroxide, nitrite, volatile organic
compounds) using the developed method here is subject to further
investigations. In addition, different exhalation modes should be
also investigated with the method in collecting EBC. Besides, the
dynamics of the air flow, mixing, and effects of temperatures and
humidity, condensation, evaporation, growth of particles during
the collection as well as the optimal straw length should be also
investigated for improving the developed technique. Overall, our
developed method here could be easily made available to a
laboratory, and have impacts on current practice of EBC
collection. Nonetheless, the reported work is a proof-of-concept
demonstration, and its performance in non-invasive disease
diagnosis such as bacterimia and virus infections needs to be
further validated including effects of those influencing factors
Figure S1 Experimental procedures used in this study
include physical characterization and molecular
analysis of the EBC collection efficiencies of the device and its
pilot application in a respiratory clinic.
Figure S3 Detection of H3N2 influenza viruses in EBC
samples collected from three human subjects with ID: 1,
2, 3 corresponding to those listed in Table S2; In
addition, spiked H3N2 virus samples were also
amplified; H3N2 viruses were used as the positive control and
DI water was used as the negative control.
Table S2 Medical conditions of seven human subjects
visiting a respiratory clinic whose exhaled breath
condensate samples were collected in this study.
PCR test and Acridine Orange stain.
Characteristics of widely used EBC collectors.
Conceived and designed the experiments: MY. Performed the experiments:
ZX FS. Analyzed the data: ZX MY. Contributed reagents/materials/
analysis tools: XL XJ YW QC. Wrote the paper: MY ZX.
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