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Journal of Breath Research
PAPER • OPEN ACCESS
Nucleic acid detection and quantitative analysis of influenza virus using
exhaled breath condensate
To cite this article: Xiaoguang Li et al 2021 J. Breath Res. 15 026001
View the article online for updates and enhancements.
This content was downloaded from IP address 46.4.80.155 on 23/03/2021 at 22:46J. Breath Res. 15 (2021) 026001 https://doi.org/10.1088/1752-7163/abd14c
Journal of Breath Research
PAPER
Nucleic acid detection and quantitative analysis of influenza virus
OPEN ACCESS
using exhaled breath condensate
RECEIVED
14 September 2020 Xiaoguang Li1,3, Minfei Wang2,3, Jing Chen1, Fei Lin1 and Wei Wang1
REVISED 1
30 November 2020 Department of Infectious Diseases, Peking University Third Hospital, Beijing 100191, People’s Republic of China
2
State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering,
ACCEPTED FOR PUBLICATION
Peking University, Beijing 100871, People’s Republic of China
7 December 2020 3
These authors contributed equally.
PUBLISHED
11 January 2021 E-mail: lixiaoguangpuh3@bjmu.edu.cn
Keywords: influenza, exhaled breath condensate, nucleic acid test, virus
Original content from
this work may be used
under the terms of the
Creative Commons
Attribution 4.0 licence.
Abstract
Any further distribution Exhaled breath condensate (EBC) is increasingly being used as a non-invasive method for disease
of this work must
maintain attribution to
diagnosis and environmental exposure assessment. We previously detected the nucleic acids of
the author(s) and the title bacterial pathogens in EBC. Influenza viruses can be transmitted through aerosols during
of the work, journal
citation and DOI. coughing and exhaling. Existing detection methods for influenza have various limitations. The
EBC collection method is convenient, non-invasive, and reduces the risk of exposure. We
investigated the detection of influenza virus in EBC using a sensitive nucleic acid testing method
and performed quantitative analysis to evaluate the present and content of influenza virus in the
breath. We evaluated 30 patients with respiratory tract infection during the 2019 influenza season.
The clinical data and samples of nasal swabs were collected for rapid influenza diagnostic (antigen)
tests. Pharyngeal swab and EBC samples were used for influenza virus nucleic acid detection. Each
EBC sample was assessed twice as well as at one-month follow-up of the patients. The nucleic acid
test in the EBC of 30 cases revealed 20 and two cases of influenza A and B, respectively, giving a
detection rate of 73.3%. The rapid influenza diagnostic (antigen) tests revealed four and 12 cases of
influenza A and B, respectively, with a detection rate of 53.3%. All pharyngeal swab samples
evaluated by the nucleic acid test were influenza-positive; 12 cases were positive for both influenza
A and B and 18 cases were positive for influenza B alone. The influenza viral load in the EBC was
103 –107 copies ml−1 . Among the 16 patients followed-up after 1 month, 4 were positive (25%) in
EBC samples and 10 were positive (62.5%) in pharyngeal swab samples. It was preliminary
exploration that influenza virus could be detected in EBC. EBC is one of the sample types that
would be used for molecular diagnosis of influenza.
1. Introduction coughing and exhaling [4]. Viable influenza A virus
was detected more often in cough aerosol particles
Exhaled breath condensate (EBC) is a promising than in exhalation aerosol particles but the difference
source of biomarkers of lung disease [1]. Since the was small because individuals’ breath more often than
year 2000, EBC has been used to detect several lung they cough. Xie et al reported that air sampling, as a
diseases such as asthma, chronic obstructive pulmon- surveillance tool for monitoring influenza activity in
ary disease, and lung cancer. Moreover, respiratory public locations, might provide early detection signals
monitoring devices are constantly being updated [2] of influenza viruses circulating in the community [5].
and the metabolomics of EBC shows the potential for Zhao et al reported that airborne transmission might
early disease diagnosis [3]. have played a role in the spread of the 2015 highly
Influenza is a common respiratory infectious dis- pathogenic avian influenza outbreaks in the United
ease that can be transmitted by respiratory droplets States [6]. In addition, during the influenza season,
between people. Lindsley et al reported that influenza health facilities are crowded, increasing the risk of
viruses can be transmitted through aerosols when influenza transmission.
© 2021 IOP Publishing LtdJ. Breath Res. 15 (2021) 026001 X Li et al
We previously conducted several studies of expir- out in accordance with the principles outlined in
atory condensate. Nanotechnology-based approaches the journal’s policy. Particularly, all investigations
such as gold nanoparticles, carbon nanotubes, and involving humans were conducted in accordance with
silicon nanowires (SiNWs) have emerged as general the principles of Declaration of Helsinki.
platforms for label-free and ultrasensitive virus detec-
tion. Among these technologies, the SiNW field effect 2.2. Research methods
transistor, when chemically modified using an anti- 2.2.1. Specimen collection
body, was shown to selectively detect single influ- 2.2.1.1. Nasal swab collection
enza viruses in liquids. We explored whether SiNW Keeping the head still, secretions were removed from
sensors can be used to detect EBC H1N1 influenza the surface of the anterior nasal orifices. We gently
virus [7]. Experimental data revealed that bacteria inserted the swab through the nasal cavity to the
and viruses in EBC can be rapidly collected using nasopharynx, held the swab for a few seconds, rotated
our developed method with an observed efficiency it gently, removed the swab, and placed it in phos-
of 100 µl EBC within 1 min. Culturing, DNA stain- phate buffer.
ing, scanning electron microscopy, and quantitative
PCR methods all detected high bacterial concentra- 2.2.1.2. Pharyngeal swab collection
tions up to 7000 CFU m−3 in EBC, including both The tongue was gently pressed with a tongue
viable and dead cells of various types. Influenza A depressor and the secretion of the palatine arch,
H3N2 virus was also detected in one EBC sample pharyngeal, and tonsil was quickly wiped on both
[8].We used a newly developed protocol of integrat- sides of the mouth of the patient with a pharyn-
ing an EBC collection device (PKU BioScreen) and geal swab while avoiding touching other parts of the
loop-mediated isothermal amplification to investig- mouth. The swab was quickly placed in the collec-
ate which bacterial pathogens are exhaled by humans, tion tube containing virus storage solution. The swab
such as Haemophilus influenzae, Pseudomonas aer- rod was broke off near the top, and the tube cover
uginosa, Escherichia coli, Staphylococcus aureus, and was tightened for sealing. The samples were stored at
methicillin-resistant S. aureus [9]. We also studied −70 ◦ C until testing.
volatile organic compounds (VOCs) in EBC, and the
differences in exhaled VOCs in patients with upper 2.2.1.3. Collection of EBC
respiratory tract infection and healthy individuals The patient was asked to exhale for 5 min. Approxim-
[10]. In this study, we focused on influenza virus in ately 500 µl EBC was collected using the breath con-
the EBC. densate collection device developed by the bioaerosol
Non-invasive and convenient testing methods are research group of Peking University [8].
needed to detect respiratory viruses, such as influenza The collection steps were as follows: (a) The cus-
virus. The EBC is non-invasive and easy sampling tomised icebox was wiped with alcohol-soaked cot-
option for children, the elderly, and people unable ton for disinfection and subjected to ultra-low tem-
to tolerate other sampling methods. However, few perature treatment (−70 ◦ C). (b) A layer of sterile
studies have examined whether influenza viruses can ultra-hydrophobic membrane, also treated at ultra-
be detected in EBC. In this study, we investigated low temperature, was placed on the sterile icebox, the
whether EBC can be used to detect influenza virus top cover was placed on the sampling box, and a sterile
using quantitative analysis. blowing pipe was inserted into the exhalation inlet.
The subjects were asked to exhale steadily and slowly
2. Methods at a normal breathing rate for 5 min. (c) After collec-
tion, the super hydrophobic membrane was removed
2.1. Research objectives in a sterile environment. The exhaled condensate
In the 2019 spring influenza season (from 5 March beads on the membrane surface were dragged back
to 8 April 2019), 31 patients were chosen from the and forth with the sterile gun head and transferred
Department of Infectious Diseases, Peking University to a sterile centrifuge tube to complete the collec-
Third Hospital; one patient was omitted because of tion process. The samples were stored at −20 ◦ C until
a lack of clinical data. Thus, 30 patients were finally testing.
enrolled in the study. The inclusion criteria were
(a) fever > 37.2 ◦ C; (b) cough and/or sore throat; 2.2.2. Reagents and testing method
and (c) age range of 18–65 years. After one month, 2.2.2.1. Rapid detection of influenza virus antigen
all patients underwent follow-up assessment, and 16 We used an influenza A/B antigen detection kit
patients agreed to the collection and re-testing of EBC (colloidal gold method) from Guangzhou Wanfu Bio-
and pharyngeal swabs. technology Co., Ltd (Guangzhou city, Guangdong
All study participants provided informed consent, Province, China). The principle is that the specific
and the study design was approved by the Peking Uni- antibody is first fixed in a certain zone of the nitro-
versity Third Hospital Medical Science Research Eth- cellulose film; when one end of the dried nitrocellu-
ics Committee 2017 (011-02). The study was carried lose is immersed in the sample, the sample will move
2J. Breath Res. 15 (2021) 026001 X Li et al
Table 1. Standard point concentration table. 2.3. Clinical data collection
Copy number of plasmids Demographic data of the patients such as gender and
Standard substance (copies ml−1 ) age, clinical data, blood routine, and influenza virus
antigen test results were collected.
E8 1.0 × 108
E7 1.0 × 107
2.4. Statistical analysis
E6 1.0 × 106
E5 1.0 × 105 Statistical analysis was performed using SPSS version
E4 1.0 × 104 17software (SPSS, Inc., Chicago, IL, USA). Data are
E3 1.0 × 103 expressed as the mean ± standard deviation (x̄ ± s).
Calculations were based on the standard curve equation. Quantities are expressed as percentages.
FAM channel: Y = −3.760865 log(X) + 48.225266
VIC channel: Y = −4.033391 log(X) + 51.186398 2.5. Safety precautions
Medical staff should adopt standard protection and
wear N95 masks, hats, isolation gowns and gloves to
forward along the film via capillary action. When ensure safety. Collecting sample is in separate rooms.
moving to the antibody region, the antigen in the There are ultraviolet disinfection devices and air puri-
sample reacts with the membrane antibody, causing fiers in the room. Medical staff should do air disinfec-
the immunocolloidal gold stain to react and develop tion after patient sampling. Laboratory staffs do the
the colour. The detailed method is as follows: nasal influenza virus nucleic acid detection in PCR labor-
swabs are collected from patients and placed into atory to ensure biosafety during test operations.
0.4 ml virus preservation solution, after which 80 µl
is removed and added to the sample chamber in the 3. Results
test card. The colour change results can be observed
within 15–20 min. 3.1. Clinical data of 30 patients
The patients’ age ranged from 18 to 57 years, with
2.2.2.2. Influenza virus nucleic acid detection a median age of 23 years. There were 15 males and
Each pharyngeal swab sample was tested once, 15 females (1:1 male-female ratio). The mean body
whereas each EBC sample was tested twice using an temperature was 38.5 ± 0.6 ◦ C (37.5–39.5 ◦ C). The
influenza A/B virus RNA detection kit (Suzhou Tian- results of blood routine examination were as follows:
long Biotechnology Co., Ltd, Suzhou city, Jiangsu white blood cell count 7.38 ± 4.49 × 109 l−1 , normal
Province, China) (the kit is referred to in this article in 26 cases, increased in two cases, decreased in one
as Tianlong). case, and absent in one case. The neutrophil percent-
age was 66.8 ± 11.9%, which was increased in nine
cases, decreased in two cases, and was absent in one
2.2.2.3. Detection method case (table 2).
2.2.2.3.1. Tianlong
The RNA of influenza A/B virus was detected using a 3.2. Pathogenic detection using nasal swab
fluorescence PCR method; 8 µl of EBC was directly and pharyngeal swab sample
added to 32 µl reaction solution and detected with The rapid influenza virus antigen detection test (col-
an ABI7500 fluorescence PCR instrument (Applied loidal gold method) using nasal swab revealed four
Biosystems, Foster City, CA, USA). Negative and pos- cases of influenza A, 12 cases of influenza B, and 14
itive results were determined by analysing the cycle negative cases. The influenza virus nucleic acid detec-
threshold (Ct) value. FAM and VIC were used as the tion using pharyngeal swab sample showed that 12
fluorescent channels for the influenza A and B probes, cases were positive for co-infection of influenza A and
respectively. The FAM channel, when the Ct ⩽37.0, influenza B and 18 cases were positive for influenza B
indicated that the influenza A virus RNA was positive, alone (table 2).
whereas the VIC channel, when the Ct ⩽37.0, indic-
ated that the influenza B virus RNA was positive. 3.3. Pathogenic detection and quantitative analysis
of influenza virus using EBC sample
2.2.2.3.2. Determination of quantitative value EBC nucleic acid detection was performed in 30
of the Tianlong kit patients. Each sample was assessed twice with Tian-
Influenza A/B plasmid was diluted to 1.0 × 109 . long reagent. The first test results showed that 15
Thereafter, it was diluted by ten-fold to six concen- cases were positive for influenza A and two positive
trations named as E8–E3, and F8–E3. These samples for influenza B. The second test results showed that
were used as standards. Table 1 shows the specific 18 cases were positive for influenza A; thus, 13 cases
concentrations. Fluorescence PCR was conducted to were positive for influenza A in both tests. A nucleic
detect the linear relationship between the Ct value acid test result that was positive once was defined as
and logarithm of the plasmid copy number, after positive. A total of 22 cases were positive, with 20 cases
which the sample concentration was determined. of influenza A and two cases of influenza B (table 2).
3Table 2. Nucleic acid test results of influenza virus in EBC of 30 patients.
Pharyngeal Pharyngeal Pharyngeal
EBC Ct EBC EBC Ct EBC swab FAM swab VIC swab
No. Sex Age T. WBC 109 L−1 N% RIDT value 1 results 1 value 2 results 2 Ct value Ct value results
1 M 24 37.5 5.73 59 — 25.62 FluA 27.58 FluA 25.98 FluB
2 F 29 39 9.13 78.2 — 29.34 FluA 30.42 FluA 28.34 FluB
4 F 23 39 5.42 65.7 — 24.24 FluA 30.72 FluA 25.23 17.66 FluA/B
5 F 57 37.5 27.45 92 — 28.32 FluA — 31.96 25.80 FluA/B
J. Breath Res. 15 (2021) 026001
6 F 38 38.7 5.65 62.5 FluA — 32.61 FluA 27.68 FluB
7 M 26 38.8 9.18 77.2 — 27.94 FluA — 25.19 FluB
8 M 23 38.7 5.48 65.8 FluB 27.48 FluA 29.29 FluA 26.86 15.09 FluA/B
9 M 23 39.1 7.8 70.9 — — — 25.60 FluB
10 M 20 37.5 7.26 67.5 — 21.70 FluA 22.91 FluA 25.85 17.55 FluA/B
11 F 22 38.7 3.22 39.1 FluB — — 31.56 22.77 FluA/B
12 F 20 38.6 4.36 62.8 FluB 19.48 FluA 22.48 FluA 28.22 19.04 FluA/B
13 M 25 39.5 4.48 58.5 — 20.53 FluA 23.34 FluA 23.60 FluB
4
14 F 20 39.3 6.9 81.7 FluA 28.43 FluA 23.32 FluA 18.55 26.14 FluA/B
15 M 23 39.5 6.2 75.7 — — — 25.42 16.77 FluA/B
16 M 23 39.3 9.17 80.1 FluB 29.57 FluA 30.01 FluA 25.97 FluB
17 F 24 38.5 5.8 72.4 FluB 35.13a FluB — 30.46 22.19 FluA/B
18 M 20 38.6 14.41 78 — — 25.03 FluA 26.13 FluB
19 M 25 39 4.86 62.7 FluB — — 25.05 FluB
20 M 19 38.5 — — FluB 25.39 FluA 25.10 FluA 26.23 FluB
21 F 22 38.5 9 76.1 — 28.58 FluA 27.54 FluA 24.65 FluB
22 M 24 38 7.5 — FluB — — 27.44 19.05 FluA/B
23 F 18 38.5 5.59 55.5 — 30.44 FluA 27.17 FluA 34.08 32.06 FluA/B
24 F 23 38 4.7 55.8 FluB — — 26.49 FluB
25 F 19 39 6.97 53.5 FluB — 33.07 FluA 19.78 FluB
26 M 31 38.3 4.32 49.6 FluA — 31.72 FluA 21.57 FluB
27 F 24 38.2 9.13 83.7 — 21.14 FluA 22.44 FluA 25.31 FluB
28 F 21 38.5 5.88 59.4 FluA — 28.30 FluA 21.32 FluB
29 F 18 37.8 8.06 71.9 FluB — — 22.10 FluB
30 M 21 37.7 3.58 55.5 — — — 36.16 FluB
31 M 26 38.3 6.89 58.6 FluB 33.32a FluB — 26.18 17.74 FluA/B
No. 1–31; No. 3 was not included in the study because of a lack of data.
FAM and VIC: reporter fluorescent channels labelled with influenza A and B probes, respectively.
a
Ct values, FAM/VIC channel tests were performed on all samples; only Ct positive data are recorded in the table. Except for No.17 and 31, the Ct value of EBC first test was positive for the VIC channel, indicating influenza B, while
the other Ct values were positive for the FAM channel, indicating influenza A.
X Li et al
WBC: white blood cell, N%: neutrophil percentage, RIDT: rapid influenza diagnostic (antigen) test, FluA: influenza A, FluB: influenza B.J. Breath Res. 15 (2021) 026001 X Li et al
As each EBC sample was tested twice, there were 16 EBC tests, 12 were negative and 4 were positive.
two Ct values with similar results; we chose the Ct The positive detection rate was reduced from an ini-
value of the first test only for further quantitative ana- tial 75% to 25% (table 5).
lysis to describe the range of concentrations at which The follow-up results of the nucleic acid test
influenza viruses can be detected in EBC. Based on the showed that among 16 pharyngeal swabs that were
results of the first test with Tianlong reagent and the initially positive, 10 were positive at follow-up. The
standard curve, quantitative detection revealed that positive detection rate decreased to 62.5%. Compar-
the influenza virus load in the EBC was 103 –107 cop- ison of the initial and follow-up results showed that
ies ml−1 (table 3). for six cases of co-infection influenza A and B at the
initial stage, one case became negative and five cases
3.4. Comparison of EBC, pharyngeal swabs, still tested positive for influenza B. For 10 cases of
and nasal swabs detection methods (table 4) influenza B at the initial stage, five became negative
The gold standard is the pharyngeal swab nucleic acid and five still showed influenza B (table 5).
test, which was compared with the nasal swab col-
loidal gold method and breath condensate nucleic 4. Discussion
acid test. The positive detection rate of influenza A/B
was 100% in the pharyngeal swab nucleic acid test, Using EBC as a sample type for influenza virus detec-
53.3% in nasal swab RIDTs, and 73.3% in the EBC tion and quantitative analysis is a novel approach.
nucleic acid test. In this study, we confirmed that influenza virus was
We next analysed the consistency between the detected in the EBC with viral loads of 103 –107 cop-
EBC and pharyngeal swabs nucleic acid test. For influ- ies ml−1 .
enza (including influenza A or B) detected by the two The EBC collection device can efficiently col-
methods, the coincidence rate was 73.3% (22/30). For lect bacteria and virus particles in exhaled breath
influenza A alone, the two methods were identical through impact and condensation, which can greatly
with seven positive cases and five negative cases, with reduce cross-contamination by using a disposable
a consistency rate of 40.0% (12/30). For influenza B super-hydrophobic membrane and exhalation tube.
alone, the two methods were identical and showed In addition, compared with commonly used exhala-
positive results in two cases, with a consistency rate tion condensers, such as the EcoScreen condenser and
of 6.7% (2/30). tube collection system, the device is less costly, simple
The consistency between the nasal swab colloidal to operate, and enables rapid testing. Thus, exhaled
gold and pharyngeal swab nucleic acid tests was condensate samples can be obtained from large num-
examined. For influenza (including influenza A or bers of patients during an influenza outbreak. This
B), the two methods showed a coincidence rate of technology has obtained a national invention patent,
53.3% (16/30). For influenza A and influenza B alone, and previous research showed that this method could
the coincidence rates of the two methods were 53.3% collect approximately 100 µl of EBC in 1–2 min.
(16/30) and 40% (12/30), respectively. In this study, we collected EBC in a safe and
In analysis of the consistency between the nasal patient-friendly manner. We did not require patients
swab colloidal gold and EBC nucleic acid tests, for to open their mouths widely to directly access the
influenza (including influenza A or B), the two meth- pharynx for sampling, avoiding nausea or even
ods showed identical results with 11 positive cases spillage. Therefore, the EBC collection method is
and three negative cases, giving a consistency rate convenient, non-invasive, repeatable, and reduces
of 46.7% (14/30). For influenza A alone, the two the risk of exposure. Furthermore, the EBC method
methods showed identical results, with four positive reduces the exposure of medical staff to patients dur-
cases and 10negative cases, giving a consistency rate of ing pharyngeal and nasopharyngeal swab collection
46.75 (14/30). For influenza B alone, the two methods and bronchoscope operations; in addition, it reduces
were identical with two positive cases and 18 negative patient exposure to the environment by eliminat-
cases, with a consistency rate of 66.7%. ing the need for open-mouth sampling in hospitals
and medical institutions. The sample type is a liquid,
3.5. Results of EBC in 16 patients who were which can be stored directly and relatively homogen-
followed up ized.
The results showed that 16 of the 30 patients with This study showed that the nucleic acid test res-
no symptoms were followed up 1 month later and ults of pharyngeal swabs were all positive. Whether
their EBC was collected. Comparison of the initial nucleic acid detection is too sensitive for accurate
and follow-up results showed that among the 10 detection remains unclear. The detected virus frag-
patients with influenza A, two cases were still pos- ments did not necessarily reflect pathogenicity and
itive and eight cases had become negative. The two infectivity. There was also a risk of false-positive
cases showing initial positive results for influenza B results. However, there is currently gold standard
had become positive for influenza A. Four patients method, and thus it is clinically important to develop
with a negative initial test were still negative. Of the viral detection methods.
5J. Breath Res. 15 (2021) 026001 X Li et al
Table 3. Quantitative detection of influenza virus in EBC of 30 patients.
No. FAM Ct VIC Ct Internal control Ct Results FAM virus load VIC virus load
1 25.62 23.66 FluA 1.0 × 106
2 29.34 23.73 FluA 1.0 × 105
4 24.24 23.34 FluA 2.4 × 106
5 28.32 23.58 FluA 2.0 × 105
6 23.99 —
7 27.94 23.89 FluA 2.5 × 105
8 27.48 24.44 FluA 3.3 × 105
9 24.70 —
10 21.70 24.31 FluA 1.1 × 107
11 24.33 —
12 19.48 24.52 FluA 4.4 × 107
13 20.53 23.68 FluA 2.3 × 107
14 28.43 24.28 FluA 1.8 × 105
15 23.70 —
16 29.57 23.47 FluA 9.1 × 104
17 35.13 24.13 FluB 9.6 × 103
18 24.25 —
19 24.25 —
20 25.39 24.02 FluA 1.2 × 106
21 28.58 24.01 FluA 1.7 × 105
22 24.39 —
23 30.44 23.75 FluA 5.4 × 104
24 23.62 —
25 23.83 —
26 24.43 —
27 21.14 24.04 FluA 1.6 × 107
28 24.31 —
29 24.22 —
30 24.64 —
31 33.32 23.65 FluB 2.7 × 104
Based on the results of the EBC first test with Tianlong reagent and the standard curve FluA: influenza A, FluB: influenza B.
Table 4. Comparison of EBC, pharyngeal swabs, and nasal swabs detection methods in 30 patients.
Pharyngeal swabs RT-PCR
Flu A/B Flu A Flu B
+ – + – + –
Nasal swabs + 16 0 1 3 12 0
RIDT (n) – 14 0 11 15 18 0
EBC + 22 0 7 13 2 0
RT-PCR (n) – 8 0 5 5 28 0
EBC can contain pathogens from throughout the type A (251 studies), and 9266 influenza type B
respiratory tract. Interestingly, in this study, EBC was patients (47 studies). The abnormal chest radiology
mainly positive for influenza A and pharyngeal swabs rates in COVID-19, influenza A, and influenza B were
were mainly positive for influenza B when the same 84%, 57%, and 33%, respectively, revealing signific-
qPCR method was used. The influenza virus was ant differences.
early onset in the upper respiratory tract and showed Interestingly, we also found that all 16 patients
downward invasion as the disease worsened. Influ- were followed up one month later with no symp-
enza A was more likely to invade the lower respir- toms, whereas10 cases (62.5%) of pharyngeal swabs
atory tract and lead to severe pneumonia. In some tested positive for influenza virus. Four cases (25%)
patients, pharyngeal swabs of the upper respiratory were positive in the EBC. During influenza season,
tract were negative, whereas the alveolar lavage fluid the virus detected during follow-up may have been
was positive. Influenza B was relatively mild and more permanently colonized in the respiratory tract, mak-
abundant in the upper respiratory tract. A recent ing the patient persistently asymptomatic, or a new
study by Pormohammad et al [11] supports this view. re-infection may have occurred. This suggests that the
They evaluated 540 articles which included 75 164 virus is also present in pharyngeal swabs and EBC
cases of COVID-19 (157 studies), 113 818 influenza in asymptomatic people recovering from infection.
6J. Breath Res. 15 (2021) 026001
Table 5. EBC nucleic acid test of16follow-up patients.
Initial EBC Follow-up EBC Initial pharyngeal swabs Follow-up pharyngeal swabs
No. FAM/VIC Ct value Results FAM/VIC Ct value Results FAM/VIC Ct value Results FAM/VIC Ct value Results
4 24.24/ FluA — 25.23/17.66 FluA/B —
7 — — /25.19 FluB /28.23 FluB
7
9 — — /25.60 FluB —
12 19.48/ FluA 25.51/ FluA 28.22/19.04 FluA/B /29.10 FluB
13 20.53/ FluA — /23.60 FluB 34.04 FluA
14 28.43/ FluA — 18.55/26.14 FluA/B /27.09 FluB
17 — 29.97/ FluA 30.46/22.19 FluA/B /29.23 FluB
18 25.03/ FluA — /26.13 FluB —
21 28.58/ FluA 21.71/ FluA /24.65 FluB —
22 — — 27.44/19.05 FluA/B /29.98 FluB
24 — — /26.49 FluB /29.63 FluB
25 — — /19.78 FluB /30.72 FluB
26 — — /21.57 FluB —
27 21.14/ FluA — /25.31 FluB —
29 — — /22.10 FluB /27.45 FluB
31 /33.32 FluB 18.28/ FluA 26.18/17.74 FluA/B /37.28 FluB
X Li et alJ. Breath Res. 15 (2021) 026001 X Li et al
Furuya-Kanamori et al [12] reported that influenza and other viruses using EBC as one of the optional
infection manifested as a wide spectrum of severity, sample types.
including symptomless pathogen carriers. A system- Techniques for EBC collection and detection
atic review and meta-analysis of 55 studies revealed are improving. McDevitt et al [19] reported the
the proportional representation of these asympto- development and performance evaluation of an
matic infected persons. The prevalence of asympto- exhaled breath bioaerosol collector for influenza
matic carriage (total absence of symptoms) ranged and evaluated its protective measures. Furthermore,
from 5.2% to 35.5% and subclinical cases (illness Fabian et al [20] optimised the detection of influ-
that did not meet the criteria for acute respiratory or enza viruses and rhinoviruses in exhaled and airborne
influenza-like illness) ranged from 25.4% to 61.8%. environments. These authors found that performing
In follow-up tests, more pharyngeal swabs than Trizol-chloroform extraction and MultiScribe™ RT
EBC samples were positive, suggesting that the col- increased virus detection by ten-fold.
onized virus was present in the pharyngeal swabs Although EBC can be used for virus detec-
or upper respiratory tract. Wang et al [13] reported tion, there are no well-developed kits, methods,
an asymptomatic couple with COVID-19; the male and gold standards, and the detection technical
patient is an intercity bus driver but did not cause indicators require further investigation. In addi-
confirmed infection of his 188 passengers. Asympto- tion, matched comparisons with other sample types
matic patients carrying viruses may only have nucleic are lacking. Thus, it is necessary to expand the
acid fragments, low pathogenicity, and low infectiv- sample size and further compare sample types to
ity. Further analysis is needed to examine these improve the detection technology. Moreover, the
points. relationship between the influenza viral load and pro-
To date, few studies have examined sing breath gnosis can be analysed according to the patients’ dis-
condensate testing for influenza viruses. George et al ease course, illness condition, and repeated detec-
[14] reported that in 19 influenza-like illnesses, influ- tion. The detection sample type used in this study
enza virus RNA was detected in nasopharyngeal showed that detection was possible for influenza
swabs and EBC with 12 positive cases in nasopharyn- and can provide a reference for studying the novel
geal swabs and one positive case in EBC. Moreover, coronavirus.
Fabian et al [15] reported four cases of influenza virus
RNA in the EBC (three were influenza A virus and 5. Conclusion
one was influenza B virus in 12 cases influenza-like ill-
nesses). Houspie et al [16] screened for 14 respiratory This preliminary exploration revealed that influenza
viruses in EBC samples from 102 upper respiratory- virus could be detected and quantified in EBC. EBC
infected people and detected six rhinoviruses and one is a useful sample type for molecular diagnosis of
influenza B virus. Costa et al [17] reported the detec- influenza. This study can serve as a helpful reference
tion of respiratory herpes virus DNA in the EBC with for further studies of other respiratory infectious dis-
only one of the 24 samples showing positive results. eases, particularly novel severe acute respiratory syn-
However, the detection rates in previous studies were drome coronavirus 2 (SARS-CoV-2).
low because of the low concentration of EBC virus.
The viral load levels found in the EBC were similar Data availability statement
to those found by traditional techniques. Granados
et al reported the use of RT-PCR for evaluation of 774 The data supporting the findings of this study are
nasopharyngeal swabs to determine the mean viral available upon reasonable request from the authors.
load (log10 copies ml−1 ) of influenza A/H3N2 (6.94)
and influenza B (4.96).This indicates that EBC is a Acknowledgments
suitable sample type for influenza detection.
Influenza detection methods such as rapid detec- The authors thank Weiwei Li and Ju Wang from
tion of influenza virus antigens have low sensitivity Shanghai Xingyao Medical Technology Development
and detection accuracy. Yarbrough et al [18] reported Co., Ltd for their support with the experiment tech-
that the sensitivity of rapid influenza diagnostic (anti- nology. The authors thank Maosheng Yao from State
gen) tests varies by up to 50%, whereas that for influ- Key Joint Laboratory of Environmental Simulation
enza B viruses is lower. The Food and Drug Admin- and Pollution Control, College of Environmental Sci-
istration reclassified rapid influenza testing devices ences and Engineering, Peking University, for his sup-
from Class I to Class II devices in 2018, requiring that port and suggestions about the study.
they must fulfil the lowest performance requirements
and comply with additional controls. However, rapid Author contributions
molecular detection technology is needed, particu-
larly for detecting nucleic acids. Further studies will Xiaoguang Li was the principal clinical investigator,
lead the development of more sensitive tests, which research designer, and thesis writer; Minfei Wang per-
can be used for influenza virus, novel coronavirus, formed experiments and collected the EBC; Jing Chen
8J. Breath Res. 15 (2021) 026001 X Li et al
was an associate clinical investigator; Fei Lin and Wei [8] Zhenqiang X, Shen F, Xiaoguang L, Yan W, Chen Q, Jie X and
Wang performed clinical tests. Yao M 2012 Molecular and microscopic analysis of bacteria
and viruses in exhaled breath collected using a simple
impaction and condensing method PloS One 7 e41137
Conflict of interest [9] Zheng Y, Chen H, Yao M and Xiaoguang L 2018 Bacterial
pathogens were detected from human exhaled breath using a
novel protocol J. Aerosol. Sci. 117 224–34
The authors declare no conflict of interest. [10] Wang J, Zheng Y, Liu Z, Yao M, Xiaoguang L, Zhuang J and
Peng W 2018 Differences in exhaled VOCs from patients
with upper respiratory tract infection and healthy ones Acta
Funding Sci. Nat. Univ. Pekin. 54 807–13
[11] Pormohammad A, Ghorbani S, Khatami A, Razizadeh M H,
This work was funded by the National Major Alborzi E, Zarei M, Idrovo J-P and Turner R J 2020
Science and Technology Projects (Project No. Comparison of influenza type A and B with COVID-19: a
global systematic review and meta-analysis on clinical,
2018ZX10732401-003) and Beijing Haidian Dis- laboratory and radiographic findings Rev. Med. Virol. e2179
trict Preventive Medicine Fund (Grant No. [12] Furuya-Kanamori L, Cox M, Milinovich G J,
2017HDPMA04). Magalhaes R J S, Mackay I M and Yakob L 2016
Heterogeneous and dynamic prevalence of asymptomatic
influenza virus infections Emerg. Infect. Dis. 22 1052–6
ORCID iD [13] Wang L, Xu X, Ruan J, Lin S, Jiang J and Ye H 2020
Quadruple therapy for asymptomatic COVID-19 infection
Xiaoguang Li https://orcid.org/0000-0002-9438- patients Expert Rev. AntiInfect. Ther. 18 617–24
5313 [14] George K S, Fuschino M E, Mokhiber K, Triner W and
Spivack S D 2010 Exhaled breath condensate appears to be
an unsuitable specimen type for the detection of influenza
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