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\begin{document}

\title{Nucleic acid detection and quantitative analysis of influenza virus
using exhaled breath condensate}



\author[1]{Xiaoguang Li}%
\author[2]{Minfei Wang}%
\author[1]{jing chen}%
\author[1]{Fei Lin}%
\author[1]{Wei Wang}%
\affil[1]{Peking University Third Hospital}%
\affil[2]{Peking University}%


\vspace{-1em}



  
  \date{May 05, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Background: Influenza is a common respiratory infectious disease that
can be transmitted by respiratory droplets from person to person. There
were few researches on influenza virus in exhaled breath. This study
aimed to investigate the detection of influenza virus in exhaled breath
condensate (EBC) and conduct quantitative analysis to evaluate the
existence and content of influenza virus in breath. Methods: We
recruited 30 patients with respiratory tract infection in the 2019
influenza season. The clinical data and samples of nasal swabs were
collected and rapid influenza diagnostic (antigen) tests (RIDTs) and EBC
influenza virus nucleic acid detection were performed. Each sample was
assessed thrice for qualitative and quantitative analysis. The patients
were followed up after one month, and the EBC was assessed again.
Results: The 30 cases were assessed for the presence of influenza virus
nucleic acid in each EBC sample three times. The results showed that
seven cases were positive for influenza A/ three times, seven cases were
positive for influenza A twice, seven cases were positive for influenza
A once, two cases were positive for influenza B once, and seven cases
were negative for all three times. Furthermore, quantitative analysis
showed that the influenza viral load in the EBC ranged from 103 to 107
copy/ml. Conclusion: The influenza virus can be detected using EBC and
the concentration can be calculated. Thus, EBC can be used for molecular
diagnosis of influenza.%
\end{abstract}%



\sloppy


\textbf{Nucleic acid detection and quantitative analysis of influenza
virus using exhaled breath condensate}

Running title: \textbf{Quantitative analysis of influenza virus with
EBC}

\par\null

Xiaoguang Li\textsuperscript{a\#}, Minfei Wang\textsuperscript{b\#},
Jing Chen\textsuperscript{a}, Fei Lin\textsuperscript{a}, Wei
Wang\textsuperscript{a}

\textsuperscript{a} Department of Infectious Diseases, Peking University
Third Hospital, Beijing 100191, People's Republic of China

\textsuperscript{b} State Key Joint Laboratory of Environmental
Simulation and Pollution Control, College of Environmental Sciences and
Engineering, Peking University, Beijing 100871, People's Republic of
China

\textsuperscript{\#}These authors contributed equally

Email addresses: Xiaoguang Li: lixiaoguangpuh3@bjmu.edu.cn, Minfei Wang:
mingfeiwang@pku.edu.cn, Jing Chen: cj850101@qq.com, Fei Lin:
leaflin66@163.com, Wei Wang: brown@bjmu.edu.cn

Correspondence should be addressed to Xiaoguang Li, Department of
Infectious Diseases, Peking University Third Hospital, Beijing 100191,
P.R. China, Tel: 86-10-13520004917, Email:lixiaoguangpuh3@bjmu.edu.cn

\textbf{Acknowledgments}

The authors thank Weiwei Li and Ju Wang from Shanghai Xingyao Medical
Technology Development Co., Ltd, for their support in experiment
technology. The authors thank Maosheng Yao from State Key Joint
Laboratory of Environmental Simulation and Pollution Control, College of
Environmental Sciences and Engineering, Peking University, for his
support and suggestions about the study.

\textbf{Abstract} :

\textbf{Background} : Influenza is a common respiratory infectious
disease that can be transmitted by respiratory droplets from person to
person. There were few researches on influenza virus in exhaled breath.
This study aimed to investigate the detection of influenza virus in
exhaled breath condensate (EBC) and conduct quantitative analysis to
evaluate the existence and content of influenza virus in breath.

\textbf{Methods} : We recruited 30 patients with respiratory tract
infection in the 2019 influenza season. The clinical data and samples of
nasal swabs were collected and rapid influenza diagnostic (antigen)
tests (RIDTs) and EBC influenza virus nucleic acid detection were
performed. Each sample was assessed thrice for qualitative and
quantitative analysis. The patients were followed up after one month,
and the EBC was assessed again.

\textbf{Results} : The 30 cases were assessed for the presence of
influenza virus nucleic acid in each EBC sample three times. The results
showed that seven cases were positive for influenza A/ three times,
seven cases were positive for influenza A twice, seven cases were
positive for influenza A once, two cases were positive for influenza B
once, and seven cases were negative for all three times. Furthermore,
quantitative analysis showed that the influenza viral load in the EBC
ranged from 10\textsuperscript{3} to 10\textsuperscript{7} copy/ml.

\textbf{Conclusion} : The influenza virus can be detected using EBC and
the concentration can be calculated. Thus, EBC can be used for molecular
diagnosis of influenza.

\textbf{Keywords} : Exhaled breath condensate; Influenza; Detection;
Molecular diagnosis



\textbf{1 Introduction}

Exhaled breath condensate (EBC) is a promising source of biomarkers of
lung disease. However, EBC is not a biomarker, but a matrix in which
biomarkers may be identified.\textsuperscript{1}Since the year 2000, EBC
has been used to detect several lung diseases, such as asthma, chronic
obstructive pulmonary disease, and lung cancer. Moreover, respiratory
monitoring devices are constantly being updated\textsuperscript{2} and
the metabolomics of EBC also shows the potential for early
diagnosis.\textsuperscript{3}

Influenza is a common respiratory infectious disease that can be
transmitted by respiratory droplets from person to person. Lindsley
reported that influenza viruses can be transmitted through aerosols when
coughing and exhaling. \textsuperscript{4} Viable influenza A virus was
detected more often in cough aerosol particles than in exhalation
aerosol particles, but the difference was not large owing to individuals
breathing more often than coughing. Xie reported that air sampling, as a
surveillance tool for monitoring influenza activity in public locations,
may provide early detection signals on influenza viruses that circulate
in the community. \textsuperscript{5} Zhao reported that airborne
transmission may have played a role in the spread of the 2015 highly
pathogenic avian influenza outbreaks in the United
States.\textsuperscript{6} In addition, during the influenza season,
health facilities are crowded, increasing the risk of influenza
transmission.

Before this study, our research team conducted several expiratory
condensate studies. Equipment development using the Silicon Nanowire
Sensor facility detected EBC H1N1 influenza
virus.\textsuperscript{7}Experimental data revealed that bacteria and
viruses in EBC can be rapidly collected using the method developed, with
an observed efficiency of 100\selectlanguage{greek}μ\selectlanguage{english}l EBC within 1 min. Culturing, DNA
staining, SEM, and qPCR methods all detected high bacterial
concentrations up to 7,000 CFU/m\textsuperscript{3} in EBC, including
both viable and dead cells of various types. Influenza A H3N2 virus was
also detected in one EBC sample.\textsuperscript{8} We used a newly
developed protocol of integrating an EBC collection device (PKU
BioScreen) and loop-mediated isothermal amplification (LAMP) to
investigate which bacterial pathogens are exhaled by humans, for example
\emph{Haemophilus influenzae} ,\emph{Pseudomonas aeruginosa} ,
\emph{Escherichia coli} ,\emph{Staphylococcus aureus} and
Methicillin-resistant\emph{Staphylococcus aureus}
(MRSA).\textsuperscript{9} We also studied volatile organic compounds
(VOCs) in EBC, and the differences in exhaled VOCs in patients with
upper respiratory tract infection and healthy
individuals.\textsuperscript{10} In this study, we focused on EBC
influenza virus.

There is a need for a non-invasive and convenient testing method for
respiratory viruses, such as the influenza virus. The EBC is
non-invasive and easy to repeat as a new sampling option for children,
the elderly, and people unable to tolerate other sampling methods.
However, little research has been conducted on the ability of EBC to
detect influenza viruses.

In this study, we investigated whether EBC can be used for the detection
of influenza virus, using quantitative analysis. We believe this study
could be a good reference for further studies on other respiratory
infectious diseases, especially the novel severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2).

\textbf{2. Research objectives and methods}

\textbf{2.1 Research objectives}

In the 2019 spring influenza season (from March 5 to April 8, 2019), 31
patients were chosen from the Department of Infectious Diseases, Peking
University Third Hospital; however, one patient was omitted (lacking
clinical data); thus 30 patients were finally enrolled for the study.
The inclusion criteria were (1) fever T\textgreater{}37.2\selectlanguage{ngerman}°C; (2) cough
and/or sore throat; and (3) age range between 18--65.After one month,
all patients had a follow up assessment, and 17 patients agreed to the
collection and re-testing of EBC.

All study participants provided informed consent, and the study design
was approved by the Peking University Third Hospital Medical Science
Research Ethics Committee 2017 (011-02).

\textbf{2.2 Research methods}

\textbf{2.2.1 Specimen collection}

\textbf{Nasal swab collection} : Keeping the head still, secretions were
removed from the surface of the anterior nasal orifices. We gently
inserted swab through nasal cavity to the nasopharynx; held for a few
seconds, rotated gently, and removed the swab to transport media
solution.

\textbf{Collection of EBC} : The patient was asked to exhale for 5 min.
Approximately 500\selectlanguage{greek}μ\selectlanguage{english}l EBC was collected using the breath condensate
collection device developed by the bioaerosol research group of Peking
University.\textsuperscript{7}

The collection steps are as follows: (1) The customised ice box is wiped
with alcohol-soaked cotton to sterilise and conduct ultra-low
temperature treatment (-70\selectlanguage{ngerman}°C). (2) A layer of sterile ultra-hydrophobic
membrane, also treated with ultra-low temperature, was placed on the
sterile ice box, the top cover of the sampling box was covered, and a
sterile blowing pipe was inserted into the exhalation inlet. The
subjects were asked to exhale steadily and slowly at a normal breathing
rate for 5 min. (3) After collection, the super hydrophobic membrane was
removed in a sterile environment. The exhaled condensate beads on the
membrane surface were dragged back and forth with the sterile gun head
and transferred to a sterile centrifuge tube to complete the collection
process. The samples were stored at -20 °C before testing.

\textbf{2.2.2 Reagent and method}

\textbf{Rapid detection of influenza virus antigen}

We used the Guangzhou Wanfu Biotechnology Co., Ltd. (Guangzhou city,
Guangdong Province, China) influenza A/B antigen detection kit
(colloidal gold method). An 80\selectlanguage{greek}μ\selectlanguage{english}L sample was added to the sample chamber
in the test card to dissolve the transfer preservation solution of the
sample to be tested; the results were observed within 15--20 min.

\textbf{Influenza virus nucleic acid detection Methods}

Each sample was assessed three times: twice using an influenza A/B virus
RNA detection kit, Suzhou Tianlong Biotechnology Co., Ltd.(Suzhou city,
Jiangsu Province, China), (called Tianlong), and once using influenza
A/B virus nucleic acid detection kit (fluorescence PCR) Jiangsu Shuoshi
Biotechnology Co., Ltd. (Taizhou city, Jiangsu Province, China), (called
Shuoshi).

\textbf{Detection method:}

\textbf{Tianlong} : The RNA of influenza A/B virus was detected using a
fluorescence PCR method; 8\selectlanguage{greek}µ\selectlanguage{english}L of EBC was directly added into 32\selectlanguage{greek}µ\selectlanguage{english}L
reaction solution and was detected using theABI7500 fluorescence PCR
instrument(Applied Biosystems, Seymour Fisher Technology (China) Co.,
Ltd, shanghai, China).Negative and positive results were determined by
analysing the cycle threshold (Ct) value. FAM and VIC were report
fluorescent channels, which labelled with influenza A and B probes,
respectively. The FAM channel, when Ct[?]37.0, means influenza A virus RNA
was positive, whereas the VIC channel, when Ct[?]37.0, means influenza B
virus RNA was positive.

\textbf{Shuoshi} : The RNA of influenza A/B virus was detected using a
fluorescence PCR method; 5\selectlanguage{greek}µ\selectlanguage{english}L EBC was directly added into 20\selectlanguage{greek}µ\selectlanguage{english}L reaction
solution and was detected using the ABI7500 fluorescence PCR instrument.
The negative and positive results were determined by analysing the Ct
value. The FAM channel, when Ct[?]34.7, means influenza A virus RNA was
positive, whereas the VIC channel, when Ct[?]34.8, means influenza B virus
RNA was positive.

\textbf{Determination of the quantitative value of the Tianlong kit:}

Influenza A/B plasmid was diluted to 1.0\selectlanguage{ngerman}×10\textsuperscript{9} as
original concentration. Thereafter, it was diluted 10times to six
gradients, which were named E8\textasciitilde{}E3, and
F8\textasciitilde{}E3. These were taken as the standard
point;\textbf{Table 1} shows the specific concentrations. Fluorescence
PCR was used to detect the linear relationship between the Ct value and
the logarithm of plasmid copy number, and the sample concentration was
determined through the experiment.

\textbf{2.3 Clinical data collection}

Demographic data such as gender and age of the patients, clinical data
of the patients, blood routine, and influenza virus antigen test results
were collected.

\textbf{2.4 Statistical analysis}

Statistical analysis was performed using SPSS 17 (SPSS Inc. Chicago).
The measured data are expressed as ± s. The counting data are expressed
in percentage.

\textbf{3 Results}

\textbf{3.1 Clinical data of 30 patients}

The patients' age ranged from 18 to 57, with a median age of 23. There
were 15 males and 15 females (1:1 male-female ratio). The mean body
temperature was 38.5±0.6°C (37.5--39.5°C). Results of blood routine
examination were WBC 7.38±4.49 ×10\textsuperscript{9}/L, normal in 26
cases, increased in two cases, decreased in one case, and absent in one
case. The neutrophil percentage was 66.8\% ±11.9\%, it increased in nine
cases, decreased in two cases, and was absent in one case. The rapid
influenza virus antigen detection test (colloidal gold method) showed
four cases of influenza A, 12 cases of influenza B, and 14 cases were
negative (See \textbf{Table 2} ).

\textbf{3.2 Detection results of EBC}

EBC nucleic acid detection was performed in 30 patients. Each sample was
assessed three times, twice with Tianlong reagent and once with Shuoshi
reagent. The results showed that seven cases were positive for influenza
A three times, seven cases were positive for A twice, seven cases were
positive for A once, two cases were positive for influenza B once, and
seven cases were negative all three times.

If one sample had two or three times positive detection of nucleic acid,
final nucleic acid test result was defined as positive, then the EBC
nucleic acid test resulted in 14 cases (14/30, 46.7\%) of influenza A.
If one-time nucleic acid test is positive, the result is defined as
positive. Further, the EBC nucleic acid test results had 21 cases of
influenza A and two cases of influenza B.

If one nucleic acid test was positive, the nucleic acid was compared
with the rapid detection of the influenza virus antigen. For influenza
A, four cases were positive, and nine cases were negative by the two
methods, with a consistent rate of 43.3\% (13/30). For influenza B, two
cases were positive, and 18 cases were negative by both methods, with a
consistent rate of 66.7\% (20/30) (See \textbf{Table 2} ).

\textbf{3.3 Quantitative detection of influenza virus in EBC}

Since each sample was tested three times, there were three Ct values
with similar results, we chose the Ct value of the first test only for
further quantitative analysis to describe the range of concentrations at
which influenza viruses can be detected in EBC. Based on the results of
the first test with Tianlong reagent and the standard curve, the
quantitative detection of the influenza virus showed that the influenza
virus load in the EBC was between 10\textsuperscript{3} and
10\textsuperscript{7} copy/ml. (See \textbf{Table 3} )

\textbf{3.4 Results of EBC in the 17 patients' follow up}

The results showed that 17 of the 30 patients with no symptoms were
followed-up 1 month later and their EBC was collected. Table 4 shows
that patients \#4, \#13, \#14, \#18, and \#27 results were negative for
influenza A in the follow up. Patients \#12 and \#21 were still
positive, \#17 went from negative to influenza A, and \#31 went from
influenza B to influenza A; eight patients remained negative.
(See\textbf{Table 4} )

\textbf{Discussion}

Using EBC as the sample type for influenza virus detection and
quantitative analysis is a novel method. In this study, we confirmed
that influenza virus could be detected by EBC, and the viral load of
influenza virus in EBC could be detected
at10\textsuperscript{3}--10\textsuperscript{7}copy/ml.

To date, there has been limited research using breath condensate testing
for influenza viruses. George\textsuperscript{11}reported that in 19
influenza-like illnesses, influenza virus RNA was detected in
nasopharyngeal swabs and EBC with 12 cases positive in nasopharyngeal
swabs and one case positive in EBC. Moreover,
Fabian\textsuperscript{12}reported four cases of influenza virus RNA in
the EBC (three were influenza A virus and one was influenza B virus in
12 cases influenza-like illnesses). Houspie\textsuperscript{13} screened
for 14 respiratory viruses in EBC samples from 102 upper respiratory
infected people and detected six rhinoviruses and one influenza B virus.
Costa\textsuperscript{14} reported EBC detected respiratory herpes virus
DNA with only 1 of the 24 samples being positive. However, the detection
rates of the studies are not high owing to the low concentration of EBC
virus. However, in this study, the patients had a relatively high viral
load of 10\textsuperscript{3}--10\textsuperscript{7} copy/ml, which
improved the detection rate of the influenza viruses in EBC, indicating
EBC was a suitable influenza detection sample type.

The influenza detection methods such as rapid detection of influenza
virus antigens have low sensitivity and affect the detection accuracy.
Yarbrough\textsuperscript{15}reportedthat the sensitivity of rapid
influenza diagnostic (antigen) tests (RIDTs) varies up to less than 50\%
and influenza B viruses are lower. This may be the reason for the low
coincidence rate between the RIDT method and EBC nucleic acid detection
in this study. The Food and Drug Administration (FDA) reclassified rapid
influenza testing devices from Class I to Class II devices in 2018,
requiring they must fulfil the lowest performance requirements and
comply with additional controls. However, developing rapid molecular
detection technology is needed and EBC will be one of the optional
sample types.

Techniques for EBC collection and detection are
improving.McDevitt\textsuperscript{16} reported development and
performance evaluation of an exhaled breath bioaerosol collector for
influenza and evaluated its protective measures. Furthermore,
Fabian\textsuperscript{17}optimised the detection of influenza viruses
and rhinoviruses in exhaled and airborne environments. These authors
found that Trizol-chloroform extraction and MultiScribe\selectlanguage{english} RT increased
virus detection by 10-fold.

In this study, we collected EBC in a safe and patient-friendly manner.
We didnot require patients to open their mouths much to look directly at
the pharynx for sampling, which can cause nausea or even spillage.
Therefore, the EBC collection method has the advantages of being
convenient, non-invasive,repeatable, and reducing the risk of exposure.
Furthermore,the EBC method reduces the exposure of medical staff to
patients during the pharyngeal and nasopharyngeal swab collection, and
bronchoscope operations; in addition, it reduces the patient exposure to
the environment by eliminating the need for open-mouth sampling in
hospitals and medical institutions.

Furthermore, we tested the same sample three times, and the results
showed a good consistency; this suggests other advantages of EBC.EBC
have a relatively high viral load. The sample, which is in liquid form,
is easy to handle, can be stored directly, homogenised, and directly
used for detection. However, nasopharyngeal swabs need to be handled and
diluted with preservation fluid, which is more complicated.

Pathogen collection of the EBC can cover the entire respiratory tract.
However, the influenza virus typically occurs in the upper respiratory
tract at the early stage and invades downwards with the progress of the
illness. Severe influenza patients can have a negative throat swab of
the upper respiratory tract but a positive alveolar lavage fluid. Thus,
for patients with a negative throat swab who do not accept bronchoscopy
and alveolar lavage fluid test, EBC provides a sample type selection
increasing the chance of virus detection. We expect that the nucleic
acid detection technology will continue to improve with detection
becoming more sensitive, proving useful for influenza virus and novel
coronavirus.

During the follow up assessments, two cases of EBC were still positive,
indicating that asymptomatic infected people may exist with influenza
virus, and the two cases of positive from negative may be new
infections. Like the novel coronavirus, in the current study,
asymptomatic infected people tested negative with multiple nucleic acid
tests, but one month later, they tested positive after re-examination.
Thus, further analysis and research are required toward asymptomatic
carriers and their infection detection.

The limitations of the study, although EBC can be used for virus
detection, there are no mature kits, methods, and gold standards, and
the detection technical indicators still need investigation. In
addition, there lacks matched comparisons with other sample types. Thus,
it is necessary to expand the sample size and make further comparison of
sample types to improve the detection technology. Moreover, the
relationship between influenza viral load and prognosis can also be
analysed according to the patients' disease course, illness condition,
and repeated detection. The detection sample type used in this study
showed that detection was possible for influenza and can provide a
reference for studying the novel coronavirus.

\textbf{Data availability}

The data will be provided to others on reasonable requests to the
corresponding author.

\textbf{Conflicts of interest}

The authors declare no conflict of interest.

\textbf{Funding statement}

The National Major Science and Technology Projects funded this project
{[}project no. 2018ZX10732401-003{]},

Beijing Haidian District Preventive Medicine Fund {[}grant no.
2017HDPMA04{]}

\textbf{Author contributions}

Xiaoguang Li is the principal clinical investigator, research designer,
and thesis writer; Minfei Wang is the main experimenter and EBC
collector; Jing Chen is an associate clinical investigator; Fei Lin
performed clinical tests; Wei Wang performed clinical tests.



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