Thirteen (19%) of the subjects tested positive for influenza using the rapid test and were asked to participate in the exhaled breath study. According to the rapid test and confirmatory PCR, 5 subjects were infected with influenza A virus and 7 subjects were infected with influenza B virus. Twelve of the 13 subjects provided exhaled breath filter samples; one subject reported feeling too fatigued and did not participate. Field blank filters were collected from two uninfected individuals. Analysis of the flow-time record supported the observation by field technicians that none of the subjects coughed during filter collection.

We detected influenza virus RNA in the exhaled breath of 4 (33%) subjects: three (60%) of the five patients infected with influenza A virus and one (14%) of the seven infected with influenza B virus. There was no correlation between nasal and throat swab influenza virus RNA concentrations and exhaled breath influenza virus RNA concentrations. Table 1 presents the demographic characteristics and symptoms of subjects, stratified according to whether they had detectable influenza virus RNA in their exhaled breath.

Concentrations in exhaled breath samples ranged from <48 to 300 influenza virus RNA copies per filter on the positive samples, corresponding to exhaled breath generation rates ranging from <3.2 to 20 influenza virus RNA copies per minute. Table 2 presents the influenza virus type, viral RNA exhalation rate, and viral RNA copies detected per well in each qPCR replicate from the exhaled breath analysis of 12 tested subjects. All influenza A virus samples were hemagglutinin type H3. Laboratory blanks and the two field blanks collected were negative.

Exhaled breath particle size and number data was obtained for 10 of the 12 subjects who provided filter samples. Data from the two remaining subjects, including the subject with the highest concentration of exhaled influenza virus RNA, could not be analyzed because of mask leaks. Across all subjects, total particle concentrations ranged from 67 to 8,500 particles per liter of air. Particle concentrations in the size selective bins ranged from 61 to 3,848 L⁻¹ (particles between 0.3 µm and <0.5 µm), 5 to 2,756 L⁻¹ (0.5 µm and <1 µm), 1 to 1,916 L⁻¹ (1 µm and <5 µm), and 0 to 9 L⁻¹ (≥5 µm). Figure 1 presents the averaged size distribution of the particles measured from all 10 subjects. On average 70% of the particles measured were between 0.3 µm and <0.5 µm, 17% between 0.5 µm and <1 µm, and 13% between 1 µm and <5 µm. Particles larger than 5 µm were rarely recorded (<0.1%). Of the 10 subjects, 50% exhaled more than 500 particles per liter of air. Two control samples were collected from asymptomatic subjects who were not infected with influenza. Both controls exhaled more than 500 particles per liter of air.

The relative importance of various modes of influenza transmission continue to be debated [3], [4]. We detected influenza virus RNA in the exhaled breath of 4 out of 12 influenza patients and found that >99% of exhaled particles were <5.0 µm in diameter. These findings regarding influenza virus RNA suggest that influenza virus may be contained in fine particles generated during tidal breathing, and add to the body of literature suggesting that fine particle aerosols may play a role in influenza transmission.

This study was conducted on a subset of subjects recruited in a randomized trial looking at the efficacy of face masks and hand hygiene to reduce influenza transmission in Hong Kong residents [41]. We recruited participants at three sites in Hong Kong, China during July through September of 2007. Subjects 12 years of age and older who presented with influenza-like illness (ILI) and were within the first 3 days of onset of symptoms were invited to participate in the study. A short questionnaire was used to record age, gender, clinical illness symptoms, medication use, medical and smoking history. Influenza-like-illness was defined as having a fever ≥37.8°C and two or more ILI symptoms (fever, cough, sore throat, runny nose, headache, malaise). Once written informed consent was obtained, nasal and throat swabs were collected and analyzed using a QuickVue Influenza A/B diagnostic test (Quidel Corp., San Diego, CA) to confirm influenza virus infection. A second set of nasal and throat swabs was collected and placed in viral transport media buffer (Earl's Balanced Salt Solution, 0.1% glucose, 0.5% bovine albumin and antibiotic) refrigerated at 2–8°C immediately after collection, stored at −20°C for up to 7 days, and then stored at −80°C until analyzed for laboratory confirmation of influenza virus infection by PCR. Only subjects who were determined to be infected with influenza A or B virus according to the rapid diagnostic test were asked to complete the exhaled breath collection. The institutional review boards of The University of Hong Kong and University of Massachusetts Lowell approved the research protocols. Subjects were compensated for their time and the inconvenience associated with participating in the study.

We collected exhaled breath from subjects using an Exhalair (Pulmatrix, Lexington, MA) a device which integrates optical particle counting technology (Airnet 310, Particle Measuring Systems, Boulder, CO) with airflow data obtained with a mass flow meter and also collects filter samples. Subjects breathed with a normal tidal pattern into an oro-nasal facemask (Hans Rudolph, Shawnee, KS) for approximately 20 minutes total. The face piece was connected to a respiratory T-valve which was equipped with a HEPA filter on the intake side to supply particle free, make-up air at very low resistance. A mass flow meter monitored inhalation and exhalation flows and the instrument computed and stored total and per breath flow and volume information. The outflow side of the T-valve was connected via tubing to first the optical particle counter and then to the filter sample collection part of the Exhalair device. The optical particle counter recorded particle counts in four size bins: 0.3 µm-<0.5, 0.5-<1 µm, 1-<5 µm and ≥5 µm. A vacuum pump pulled air through the tubing at 28.3 lpm into a real-time particle counting system during the exhaled breath particle characterization phase of the test (approximately 5 minutes). After completion of the particle counts, the outflow of the T-valve was attached to a 37-mm, 2-µm pore-size Teflon filter with a polymethylpentene (PMP) support ring (Pall Life Sciences, New York) and the vacuum pump was switched to pull through the filter during the particle collection phase of the test (15 minutes). Teflon filters were refrigerated at 2–8°C immediately after collection, stored at −20°C for up to 7 days, and then stored at −80°C until analyzed. T-valves and HEPA filters were disposed of after each use and masks were disinfected using 10% bleach and autoclaved prior to reuse.

Influenza virus RNA collected from the exhaled breath on the Teflon filters was extracted using a Trizol-chloroform based method modified from a protocol developed for extraction of nasal swab and lavage samples [42]. Initially, the PMP support ring surrounding the Teflon filter was cut 6 to 8 times around the circumference of the filter and the filter was put into a 50-ml polypropylene centrifuge tube containing 400 µl of phosphate buffered saline (PBS). A 10 µl mixture containing 20 µg glycogen (Ambion, Austin, TX), 15 µg glycoblue (Ambion, Austin, TX), 50 ng of Human DNA (BD Biosciences, San Jose, CA) in PBS was added directly to each sample. Next, 750 µl of Trizol LS (Invitrogen, Carlsbad, CA) was added, the sample vortexed for 30 seconds and the liquid transferred to a 1.7 ml low protein binding tube (LoBind, Eppendorf, Westbury, NY). The tubes were placed on an MS2 Minshaker (IKA works, Guangzhou, China) for 10 minutes at 300 rpm. After mixing, 230 µl of chloroform (Sigma-Aldrich, St. Louis, MO) was added and samples were briefly vortexed and placed on the shaker at 300 rpm an additional 5 minutes. Samples were then centrifuged at 2100×g for 5 minutes and 700 µl of the aqueous supernatant was transferred to a tube containing 600 µl of 100% isopropanol (Sigma-Aldrich, St. Louis, MO) for RNA precipitation. After incubating for1 hour at room temperature, RNA was pelleted by centrifuging for 12 minutes at 20000×g at room temperature. The supernatant was decanted and 600 µl of 75% ethanol were added to wash the pellets. The RNA pellets were centrifuged for 5 minutes at 20000×g, the ethanol decanted and the pellets air dried for approximately 20 minutes. The RNA was suspended in 20 µl of nuclease-free water (Promega, Fitchburg, WI) and immediately converted to cDNA.

RNA in the nasal and throat swabs was extracted from 140 µl of viral transport media using Qiagen QIAamp Viral RNA mini columns (Qiagen Corp., Valencia, CA) in accordance with the manufacturer instructions. The RNA was eluted in 100 µl of carrier buffer and immediately converted to cDNA. Unused RNA was stored at −80°C.

cDNA was synthesized from purified and concentrated RNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). A 20 µl total reaction volume was made with 10 µl RNA, 2 µl 10X RT buffer, 0.8 µl dNTP Mix (100 mM), 2.0 µl 10X RT random hexamer primers, 1.0 µl MultiScribe™ reverse transcriptase, 1 µl RNase inhibitor and 3.2 µl nuclease-free water. Synthesis was carried out in an ABI 9700 Thermocycler (Applied Biosystems, Foster City, CA) and reaction conditions were 25°C for 10 minutes, 37°C for 120 minutes, and 85°C for 5 seconds. cDNA samples were stored at −20°C.

Quantitative PCR was performed using an Applied Biosystems Prism 7500 detection system (Foster City, CA). Triplicate cDNA samples were analyzed in a 96-well plate with an adhesive film cover (Applied Biosystems, Foster City, CA). Each well contained 5 µl of cDNA template, 12.5 µl of 2X Taqman™ Universal PCR Master Mix (Applied Biosystems, Foster City, CA), 900 nM of each primer and 100 nM probe.

We tested three primer/probe sets on 17 nasal swab samples collected for this project in order to select the most sensitive for the exhaled breath and remaining nasal swab samples: a set used by the Centers for Disease Control, a set used at the Queen Mary Hospital Virology laboratory (QMH) and a set from the published literature [38]. Based on the sensitivity results from these tests the following set of primers and probe were selected to analyze the exhaled breath samples: for influenza A virus two forward primers 5′- GGA CTG CAG CGT AGA CGC TT-3′ and 5′- CAT CCT GTT GTA TAT GAG GCC CAT-3′, reverse primer 5′- CAT TCT GTT GTA TAT GAG GCC CAT- 3′, and probe 5′FAM- CTC AGT TAT TCT GCT GGT GCA CTT GCC A -3′TAMRA; for influenza B virus forward primer 5′- AAA TAC GGT GGA TTA AAT AAA AGC AA-3′, reverse primer 5′- CCA GCA ATA GCT CCG AAG AAA -3′, and probe 5′FAM CAC CCA TAT TGG GCA ATT TCC TAT GGC -3′TAMRA [38]. The primers and probe targeted the matrix (M) gene and the hemagglutinin (HA) gene for influenza A and B viruses respectively. Primers and probe were manufactured by Sigma-Proligo (Proligo Singapore Pty Ltd).

We constructed standard curves for the qPCR by making 1∶10 dilutions of cDNA made from QMH influenza A and B virus stocks. Influenza A/PR/8/34 and influenza B/Hong Kong/AE34/2002 virus stocks were grown at QMH on MDCK cells and purified via a sucrose density gradient. Once purified, 50 µl aliquots of each virus were extracted using the Trizol-chloroform method described previously, synthesized to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and quantified by qPCR with a plasmid standard curve for each influenza virus. The virus concentrations calculated based on this method were 1.88×10⁷ and 1.66×10⁶ virus particles per µl for the influenza A and B virus stocks, respectively. The limit of quantification for the qPCR was 6 influenza A or B viral RNA particles per PCR well, with all three replicates crossing the qPCR fluorescence threshold within 37 cycles. For the exhaled breath filters and nasal swabs, samples were considered positive if at least one of the three replicates crossed the qPCR cycle threshold, but were only quantifiable if all three replicates crossed the cycle threshold. Because 25% of the cDNA was used per well and 50% of the extracted RNA was used to make cDNA, the total number of virus copies per filter was 8 times the average copy number per well.

The RNA from the nasal swab samples positive for influenza A virus were shipped to the Centers for Disease Control Virus Surveillance and Diagnostics Branch Influenza Division. Samples were tested for H1 and H3 sub-types using quantitative PCR methods.

Exhaled virus concentrations in exhaled breath were computed from the qPCR results and the Exhalair record of total exhaled volume during filter sample collection. Total particle concentrations and concentrations in each size bin were calculated for each breath by dividing the number of particles counted by the volume exhaled. We then averaged the particle concentrations over all breaths collected for each subject. Computations were performed in SAS for Windows (version 9.1.3, Cary, NC).