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Influenza Virus Aerosols in Human Exhaled Breath: Particle Size, Culturability, and Effect of Surgical Masks

Influenza Virus Aerosols in Human Exhaled Breath: Particle Size, Culturability, and Effect of... The CDC recommends that healthcare settings provide influenza patients with facemasks as a means of reducing transmission to staff and other patients, and a recent report suggested that surgical masks can capture influenza virus in large droplet spray. However, there is minimal data on influenza virus aerosol shedding, the infectiousness of exhaled aerosols, and none on the impact of facemasks on viral aerosol shedding from patients with seasonal influenza. We collected samples of exhaled particles (one with and one without a facemask) in two size fractions (‘‘coarse’’.5 mm, ‘‘fine’’#5 mm) from 37 volunteers within 5 days of seasonal influenza onset, measured viral copy number using quantitative RT-PCR, and tested the fine-particle fraction for culturable virus. Fine particles contained 8.8 (95% CI 4.1 to 19) fold more viral copies than did coarse particles. Surgical masks reduced viral copy numbers in the fine fraction by 2.8 fold (95% CI 1.5 to 5.2) and in the coarse fraction by 25 fold (95% CI 3.5 to 180). Overall, masks produced a 3.4 fold (95% CI 1.8 to 6.3) reduction in viral aerosol shedding. Correlations between nasopharyngeal swab and the aerosol fraction copy numbers were weak (r = 0.17, coarse; r = 0.29, fine fraction). Copy numbers in exhaled breath declined rapidly with day after onset of illness. Two subjects with the highest copy numbers gave culture positive fine particle samples. Surgical masks worn by patients reduce aerosols shedding of virus. The abundance of viral copies in fine particle aerosols and evidence for their infectiousness suggests an important role in seasonal influenza transmission. Monitoring exhaled virus aerosols will be important for validation of experimental transmission studies in humans. Citation: Milton DK, Fabian MP, Cowling BJ, Grantham ML, McDevitt JJ (2013) Influenza Virus Aerosols in Human Exhaled Breath: Particle Size, Culturability, and Effect of Surgical Masks. PLoS Pathog 9(3): e1003205. doi:10.1371/journal.ppat.1003205 Editor: Ron A. M. Fouchier, Erasmus Medical Center, Netherlands Received October 21, 2012; Accepted January 9, 2013; Published March 7, 2013 Copyright:  2013 Milton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by the Centers for Disease Control and Prevention cooperative agreements 1U01CI000446 and 1U01IP000497, NIH grant RC1AI086900, and the U.S. Federal Aviation Administration (FAA) Office of Aerospace Medicine through the Air Transportation Center of Excellence for Airliner Cabin Environment Research (Cooperative Agreement 04-C-ACE-HU and 07-C-RITE-HU) via a subcontract from Auburn University (#06-ACER-207814). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the funding agency. Competing Interests: BJC has received research funding from MedImmune Inc., and consults for Crucell MV. The authors declare that no other competing interests exist. This does not alter our adherence to all PLoS Pathogens policies on sharing data and materials. * E-mail: dmilton@umd.edu . These authors contributed equally to this work. possible routes of infection, including use of fit-tested N95 Introduction respirators [3]. A year after the 2009 pandemic, there was no Transmission of influenza virus between humans may occur by greater clarity on the importance of the various modes of three routes: (1) direct or indirect contact between an infected and transmission [9]. a susceptible person, usually resulting in contamination of a The U.S. Centers for Disease Control and Prevention recently susceptible person’s hands followed by hand to respiratory mucosa funded an experimental study of person-to-person transmission to contact; (2) large droplet spray wherein droplets of respiratory fluid address this important knowledge gap [10]. However, an greater than approximately 100 mm in diameter are expelled with experimental study using intranasal inoculation to infect experi- sufficient momentum to deliver a direct hit on the respiratory mental donors [11] will need to show that the donors and naturally mucosa; and (3) aerosols generated by release of smaller, virus- infected persons shed similar virus aerosols with regard to containing droplets, as may occur during tidal breathing and quantity, particle size distribution, and infectiousness, given that coughing [1,2], that rapidly evaporate into residual particles earlier experiments suggested that intranasal inoculation requires (droplet nuclei),which are inhaled and deposited in the respiratory quantitatively larger doses and produces qualitatively milder illness tract [3–6]. There is significant evidence for each of these routes than does inoculation via aerosol [12]. [7,8], but their relative importance is not known [3]. As a result, In an occupational hygiene context, personal protection is the Institute of Medicine recommended that healthcare workers in usually the last resort, after source mitigation and environmental contact with 2009-H1N1 patients use protection against all of the controls are exhausted [13]. Thus, it is worthwhile considering PLOS Pathogens | www.plospathogens.org 1 March 2013 | Volume 9 | Issue 3 | e1003205 Influenza Virus Aerosols in Human Exhaled Breath Author Summary Table 1. Participant’s sex, symptoms, temperature, and influenza virus type. The relative importance of direct and indirect contact, large droplet spray, and aerosols as modes of influenza transmission is not known but is important in devising N Percent effective interventions. Surgical facemasks worn by pa- Number with complete data 37 100 tients are recommended by the CDC as a means of reducing the spread of influenza in healthcare facilities. We Male 30 81 sought to determine the total number of viral RNA copies a On antiviral medicine 00 present in exhaled breath and cough aerosols, whether the Asthmatic 514 RNA copies in fine particle aerosols represent infectious Flu shot this season 13 virus, and whether surgical facemasks reduce the amount of virus shed into aerosols by people infected with Flu shot previous seasons 12 32 seasonal influenza viruses. We found that total viral copies a Current smoker 924 detected by molecular methods were 8.8 times more Tachypnea 13 35 numerous in fine (#5 mm) than in coarse (.5 mm) aerosol Breathing difficulty 16 43 particles and that the fine particles from cases with the highest total number of viral RNA copies contained Lymphadenopathy 18 49 infectious virus. Surgical masks reduced the overall a Feverish 19 51 number of RNA copies by 3.4 fold. These results suggest Temperature $37.8uC10 27 an important role for aerosols in transmission of influenza Type A 21 57 virus and that surgical facemasks worn by infected persons are potentially an effective means of limiting the spread of Self-reported. influenza. At time of exhaled breath measurement. doi:10.1371/journal.ppat.1003205.t001 whether surgical facemasks could be effective as a means of source copies (Figure 1) was below the limit of detection with and without th control. The CDC recommends that persons with influenza wear facemasks; the 75 percentile dropped from 37 to below the limit surgical masks when in contact with susceptible individuals of detection with use of surgical masks. Using Tobit analysis, we [14,15]. However, there is only one report studying mask impact estimated that the geometric mean coarse fraction copy number on containment of infectious large droplet spray during influenza without a facemask was 12 (95% confidence interval (CI), 4 to 37) infection [16], and no data on surgical mask impact on release of and that the effect of facemasks was to produce a statistically infectious viral aerosols. significant 25 fold reduction in the copy number (95% CI 3.5 to In the current study of patients infected with seasonal influenza, 180, p = 0.002) to ,0.5 copies per 30 min sample. we describe the number of copies of viral RNA in two aerosol size We detected viral RNA in 78% (29 of 37) of fine particle fractions, report the culturability of virus in the fine-particle samples collected from volunteers when they were wearing a mask fraction, and the effect of surgical masks. and in 92% (34 of 37) of samples collected when they were not wearing a mask. Thus, the relative risk for any virus detection with mask versus without a mask was 0.85 and borderline statistically Results significant (CI 0.72 to 1.01; McNemar’s test p = 0.06). However, We screened 89 volunteers: 33 (37%) tested positive for the reduction in copy number was statistically significant: The influenza using the rapid test (20 influenza A and 13 influenza median number of viral copies in the fine particle fraction was 250 B) and were asked to provide exhaled breath samples. Eight with masks and 560 without masks. The geometric mean copy additional volunteers with negative rapid tests who reported a number in the fine particle fraction without a facemask was 110 cough and who had a temperature of $37.8uC were also invited to (95% CI 45 to 260) and the facemasks produced a 2.8 fold participate. In total, 38 volunteers were confirmed to have reduction in copy number (95% CI 1.5 to 5.2, p = 0.001). influenza virus infection by PCR of nasopharyngeal specimens. Combining the coarse and fine fractions, we detected viral RNA Exhaled breath data with and without a surgical mask are in 29 (78%) subjects when wearing facemasks and 35 (95%) when complete for 37 of the 38 volunteers (21 influenza A, 16 influenza not wearing facemasks (McNemar’s test p = 0.01). Surgical masks B); data for one volunteer has been excluded due to laboratory produced a 3.4 (95% CI 1.8 to 6.3) fold reduction in viral copies in error in sample processing. One of the infected subjects reported exhaled breath. receiving influenza vaccine for the current year. None of the Fine fraction copy numbers were on average 8.8 (95% CI 4.1 to subjects sneezed during the sample collection. Table 1 shows the 19) times larger than coarse fraction copy numbers. The coarse sex, symptom and fever prevalence, and influenza virus type and and fine fraction copy numbers were correlated (r = 0.60, Table 2 shows descriptive statistics for age and viral RNA copy p,0.0001). The viral load in the nasopharyngeal swab specimen, number in swabs and exhaled aerosol fractions of the 37 however, was not correlated with that in the coarse fraction volunteers with confirmed influenza infection. The viral copy (r = 0.17, p = 0.31) and only weakly with that in the fine fraction numbers in each of the five specimens for all 37 cases are shown in (r = 0.29, p = 0.08). There was no significant difference in copy Table S1. number between influenza A and B virus infection in either the We detected influenza virus RNA in the coarse fraction coarse (p = 0.28) or fine (p = 0.26) fraction. Reported asthma (particles greater than 5 mm) collected from 11% (4 of 37 (p = 0.029) and feverishness (p = 0.014) were associated with volunteers) while wearing surgical masks and from 43% (16 of significantly lower fine fraction copy numbers. However, coarse 37) while not wearing a mask (relative risk for virus detection with fraction copy numbers were not significantly impacted and mask = 0.25, 95% confidence interval (CI) 0.09 to 0.67; McNe- temperature measured at the time of testing was not associated mar’s test p = 0.003). The median number of coarse fraction viral with exhaled copy numbers. Vaccination in any prior year was PLOS Pathogens | www.plospathogens.org 2 March 2013 | Volume 9 | Issue 3 | e1003205 Influenza Virus Aerosols in Human Exhaled Breath Table 2. Descriptive statistics. Percentiles th th Min 25 Median 75 Max Age 18 18 19 20 54 Days since onset 01 235 3 4 5 6 7 Nasopharyngeal swab copy number 1.7610 8.3610 4.2610 1.8610 3.4610 Coarse particle copy number with mask 0 0 0 0 7.7610 1 4 Coarse particle copy number no mask 0 0 0 3.7610 2.9610 1 2 4 Fine particle copy number with mask 0 5 2.2610 2.5610 2.4610 1 2 2 5 Fine particle copy number no mask 0 1.1610 1.1610 5.6610 1.3610 At time of exhaled breath measurement. doi:10.1371/journal.ppat.1003205.t002 associated with a non-significant trend toward lower copy numbers report by Johnson et al [16], who detected influenza virus RNA in in coarse (p = 0.11) and fine fractions (p = 0.15); there were too few cough generated large droplet spray from 100% of influenza having received the current season’s vaccine to analyze. Self- patients over two brief sampling trials, and from 78% on each trial. reported tachypnea, breathing difficulty, smoking, and lymphade- These discrepant findings are likely due to the very different nopathy were not associated with significant shifts in exhaled copy collection techniques and particle sizes collected in these two studies. We used a specially designed aerosol sampler to collect numbers. We recovered infectious virus from fine particle samples (with particles from 0.05 to 50 mm in diameter. Johnson et al, by contrast, used simple deposition on petri dishes, and based on and without mask) produced by the two subjects with the highest particle settling rates and collection times, that method would have numbers of viral RNA copies in the fine particle fraction after blind passage on MDCK cells. Sequence analysis showed that the been unlikely to collect particles with diameters of less than approximately 50 mm because smaller particles would have two isolates were seasonal H1N1, with sequence differences from remained suspended in air and flowed around the petri dishes. each other and unrelated to any viruses present in the Veterinary Medicine laboratories at the time these samples were cultured. We view results from Johnson et al and the present study as complementary. Together the studies show that surgical masks Virus copy number (Table 3) declined with time since onset of can limit the emission of large droplet spray and aerosol droplets symptoms. In the coarse fraction, each additional day after onset larger than 5 mm [16]. However, surgical masks are not as was associated with a 6.0 fold drop in the number of virus copies efficient at preventing release of very small particles. It is well detected (95% CI 1.7 to 21 fold). Fine particles also declined with known that surgical masks are not effective for preventing time, each additional day after onset was associated with a 2.4 fold exposure to fine particles when worn as personal protection [18]. drop in the number of copies detected (95% CI 1.1 to 5.1 fold). We had hypothesized that when used as source control, exhaled droplets might be large enough prior to evaporation to be Discussion effectively captured, primarily through impaction. This appears We measured exhaled influenza viral particle copy number by to be true for virus carried in coarse particles. But the majority of quantitative RT- PCR in two particle size fractions, $5 mm virus in the exhaled aerosol appear to be in the fine fraction that (coarse) and ,5 mm (fine), and assayed the fine fraction for is not well contained. Nevertheless, the overall 3.4 fold reduction culturable virus. We observed that viral copy numbers were in aerosol copy numbers we observed combined with a nearly greater in the fine than in the coarse fraction, and recovered complete elimination of large droplet spray demonstrated by infectious virus from the fine particle fraction collected from the Johnson et al. suggests that surgical masks worn by infected two samples with the highest RNA copy numbers. These results, persons could have a clinically significant impact on transmission. combined with older data suggesting that the infectious dose via For example if one hypothesized that all transmission were due to aerosol is about two orders of magnitude lower than via large aerosol particles ,50 mm, and estimated a reproductive number droplets [12], suggest an important role for aerosols in seasonal of 1.5 for influenza (i.e. each infection generates 1.5 new influenza transmission. infections on average at the start of the epidemic) [19], then the Surgical masks nearly eliminated viral RNA detection in the use of surgical masks by every infected case could reduce the coarse aerosol fraction with a 25 fold reduction in the number of reproductive number below 1 [20]. Compliance, however, would viral copies, a statistically significant 2.8 fold reduction in copies be a major limitation resulting in lower efficacy in real-world detected in the fine aerosol fraction, and an overall statistically practice [21,22]. significant 3.4 fold reduction of viral copy number in the exhaled While it is generally assumed that large droplets shed from the aerosols. This finding supports current Centers for Disease Control respiratory tract contain infectious virus, there are limited data and Prevention recommendations that healthcare facilities en- that indicate that fine particle aerosols released from the human courage patients with influenza-like illness to don surgical respiratory tract contain infectious virus. In one previous study by facemasks as one component of an influenza infection control Lindsley et al, infectious virus was detected in 2 of 21 cough program [17]. aerosol samples, once with a sampler that did not discriminate When volunteers were not wearing surgical masks, we detected between coarse and fine particles and once in the coarse particle virus RNA in coarse particles exhaled by 43% and in fine particles fraction of a second instrument [23]. This observation, along with exhaled by 92% of influenza patients. This is in contrast to the our observation that it was possible to recover culturable virus PLOS Pathogens | www.plospathogens.org 3 March 2013 | Volume 9 | Issue 3 | e1003205 Influenza Virus Aerosols in Human Exhaled Breath Figure 1. Influenza virus copy number in aerosol particles exhaled by patients with and without wearing of an ear-loop surgical mask. Counts below the limit of detection are represented as 0.5 on the log scale. doi:10.1371/journal.ppat.1003205.g001 from the fine-particle fraction using our device demonstrates that viral RNA) represents technical difficulties in sampling and humans generate infectious influenza aerosols in both coarse and culturing exhaled breath samples or whether the vast majority of fine particle fractions. This lends support to the hypothesis that the virus exhaled by influenza A patients is actually non-infectious. aerosols may be a common pathway for influenza transmission These findings are consistent with those by Lindsley et al. [23] We among humans [8,24]. However, a clear test of the hypothesis designed the sampler specifically to overcome problems with requires intervention studies that can interrupt only one mode of existing bioaerosol samplers, including efficiently collecting sub- transmission without interfering with others [25]. micron particles into a liquid and use of appropriate buffer to preserve infectiousness [27]. We have previously shown that We only detected infectious virus in exhaled breath samples 4 5 with high (10 to 10 ) copy numbers by quantitative RT-PCR. collection on solid, dry collection media resulted in large losses of culturability [26]. Therefore, we did not attempt to culture the This implies that the ratio of total viral particles to infectious virus 3 4 2 3 was about 10 to 10 , compared with 10 to 10 for laboratory coarse fraction collected on a Teflon substrate. Subsequent studies stocks and experimental aerosols [26]. It is not yet known whether in our laboratory indicated that about 50% of the infectious virus the low recovery of infectious virus (despite high copy numbers of is lost during the concentration step of our procedure (data not PLOS Pathogens | www.plospathogens.org 4 March 2013 | Volume 9 | Issue 3 | e1003205 Influenza Virus Aerosols in Human Exhaled Breath Table 3. Copy number coarse and fine exhaled particles without surgical mask by day since onset of influenza symptoms. Number of Virus Particles Days Since Onset Particle Size Number of Cases Min Median Maximum 4 6 7 1Swab 10 2.1610 1.1610 3.4610 1 4 Coarse ,LD 2.3610 2.9610 2 5 Fine 4 6.1610 1.3610 4 5 6 2Swab 15 1.7610 1.0610 3.4610 Coarse ,LD ,LD 4.7610 1 4 Fine ,LD 2.1610 3.9610 4 6 7 3 Swab 7 2.3610 1.4610 1.0610 Coarse ,LD ,LD 1.1610 1 2 Fine 2 3.7610 5.3610 4 5 6 4 Swab 3 8.1610 4.2610 1.5610 Coarse ,LD ,LD ,LD 1 1 2 Fine 3.2610 7.5610 4.4610 Because there were only single cases studied on day 0 (day of onset) and on day 5 since onset of symptoms, only data for cases studied on days 1 through 4 after onset of symptoms are shown. doi:10.1371/journal.ppat.1003205.t003 shown), suggesting that this is one contributing factor in the low significant inter-individual variation and modeling suggests that rate of recovery of infectious virus in this study. cases with higher viral loads are disproportionately important in the spread of influenza [30,31]. Additional studies are also needed The lack of strong correlation between the viral load in the nasopharyngeal and aerosol samples is possibly of interest. This to determine how aerosol generation correlates with symptoms may merely be a result of nasopharyngeal sample variability; in (including milder disease), presence of other health conditions, age (we studied a narrow age distribution), and co-infection with other future studies, control for sample quality by PCR of a cellular gene may be helpful. Our sampler, as is the case with all samplers for respiratory viruses so that recommendations for infection control fine and ultrafine particles, has an upper limit to the size droplet can be critically evaluated. that can be pulled into its inlet airstream. Thus, a second possible explanation for the lack of correlation is that the nasopharynx is Methods primarily a source for very large droplets (.50 mm) that we would Patient population not have detected. Furthermore, none of our subjects sneezed; an We recruited volunteers with influenza-like illness from the Lowell, efficient method of generating droplets from the upper respiratory MA community, primarily among students and staff of the University tract. This may imply that the smaller droplets we detected were of Massachusetts, beginning January 29 and ending March 12, 2009. generated in the lower respiratory tract and that the viral load at The study protocol was approved by the Institutional Review Boards that location is not strongly correlated with the nasopharyngeal of the University of Massachusetts Lowell, Lowell General Hospital, load. Alternatively, shedding into aerosol droplets may be driven and Saints Memorial Hospital, Lowell, MA. Oral informed consent by other host factors (e.g. asthma, symptom severity, and immune was obtained by providing each subject with a detailed consent response), co-infection with other agents, virus factors affecting information form. Collection of a signed copy of the form was waived release from the epithelium, or the nature of the resident because it would have been the only personally identifiable microbiome. If shedding into aerosol is determined in large part information retained by this minimal risk study. by the location of infection in the respiratory tract, this may have Volunteers learned of the study through flyers and notices implications for experimental studies of transmission [11,28]. Such posted on campus and by referral from health care providers. We studies will need to monitor aerosol shedding to determine screened self-referred volunteers by telephone for influenza-like whether nasal inoculation of donors results in aerosol shedding illness (ILI). Persons who reported onset of fever and cough within that mimics naturally acquired infection to validate the experi- the preceding 72 hours or were referred by a health care provider mental design and aid the interpretation of results. were invited to the laboratory for testing. We collected a Most of the viral aerosol generation we observed occurred nasopharyngeal specimen using a flocked swab (501CS01, Copan during the first days of symptomatic illness (Table 3), consistent Diagnostics, Murrieta, CA) and temperature was taken with a with studies of shedding monitored by nasal washes [29]. We digital ear thermometer (Model 18-200-000, Mabis Healthcare, studied each individual on only one occasion and, by design, have Waukegan, IL). All volunteers with a temperature $37.8uC and a little data beyond day 3. Further longitudinal studies of viral cough and volunteers without fever who provided a nasopharyn- aerosol generation are needed to confirm these findings. New geal specimen positive for influenza by point of care testing study designs will be needed to examine aerosol generation before (QuikVue Influenza A/B, Quidel Corp., San Diego, CA) were and on the day of symptom onset in community acquired invited to provide exhaled breath samples, answer a questionnaire, infection. A limitation of our study is that we recruited patients and provide a second nasopharyngeal specimen for analysis by with certain signs and symptoms or who were positive on a rapid PCR. Only subjects with influenza infection confirmed by PCR test or had fever, and therefore our data could be biased towards were included in the data analysis. patients with higher viral loads [21]. However, we still observed PLOS Pathogens | www.plospathogens.org 5 March 2013 | Volume 9 | Issue 3 | e1003205 Influenza Virus Aerosols in Human Exhaled Breath Exhaled breath collection We collected exhaled breath with the subject seated in front of the inlet for a sampler designed for human exhaled breath collection, Figure 2, (G-II) described in detail by McDevitt et al. [27] Briefly, the G-II inlet was cone shaped so that the subject’s face was situated inside the large end of an open cone with air drawn continuously around the subject and into the sampler. The cone allows the subject to breathe normally and unlike use of a mouthpiece, the subject could also wear a mask. The cone served as a capture type ventilation hood allowing collection of exhaled breath with minimal fugitive emissions even when the subject was wearing a mask with resultant redirection of flow. Intake air (130 L/min) flowed through a conventional slit impactor that collected particles larger than 5 mm on a Teflon surface (‘‘coarse’’ particle fraction). To collect a ‘‘fine’’ particle fraction, water vapor was condensed on the remaining particles, which created droplets large enough to be captured by a 1.0-mmslit impactor. The 1.0-mm impactor was composed of a slit and a steel impaction surface sealed inside a large reservoir. Impacted droplets drained from the impaction surface into a buffer- containing liquid in the bottom of the reservoir. Concentrated buffer was pumped into the reservoir during collection to match the accumulation of water from collected droplets and maintain phosphate buffered saline with 0.1% bovine serum albumin throughout collection. The sampler was shown to be 85% efficient for particles greater than 50 nm in diameter and was comparable to the SKC BioSampler for detection and recovery of influenza A/PR/8/34 H1N1 by PCR and culture. Between subjects, the apparatus was disassembled and cleaned with a 0.5% hypochlorite solution. Exhaled particles were collected for 30 minutes while the subject wore an ear-loop surgical mask (Kimberly-Clark, Roswell, GA) and then for 30 minutes without a mask. Subjects were asked to cough 10 times at approximately 10-minute intervals for a total of 30 coughs during each 30 minute sample. One subject coughed frequently such that forced coughs were not required. No subjects Figure 2. Exhaled breath collection system. Each volunteer sat as shown with face inside the inlet cone of the human exhaled breath air were observed to sneeze. sampler inside a booth supplied with HEPA filtered, humidified air for 30 min while wearing an ear-loop surgical mask. Three times during the Sample analysis 30 min each subject was asked to cough 10 times. After investigators Immediately after collection, the Teflon impaction surface was changed the collection media, the volunteer sat in the cone again, without wearing a surgical mask, for another 30 min with coughing as removed and temporarily stored at 220uC. The impactors were before. scraped with a flocked swab wetted with Dulbecco’s phosphate doi:10.1371/journal.ppat.1003205.g002 buffered saline with calcium and magnesium (Hyclone, Thermo Scientific, Waltham, MA) with 0.1% bovine serum albumin 3.0610 virus particles per mL or a stock of influenza B (B/Lee/ (DPBS++BSA). The swab was eluted in 600 ml of DPBS++BSA for 1940, Advanced Biotechnologies Incorporated, Columbia, MD) 1 minute with vortexing. The resulting sample was stored at with a concentration of 8.6610 virus particles per mL as 280uC. determined by electron microscopy. Results are expressed as the The fine particle fraction collected in DPBS++BSA buffer (100 total number of virus particles by reference to the standard curve, to 150 ml volume) was maintained at 4uC and concentrated by rounded to the closest integer value. The limits of detection were 6 ultrafiltration using Amicon Ultra 15 filter units with a molecular and 11 viral RNA copies per qPCR well for influenza A and B weight cut off of 100 kD (Millipore, Bedford, MA) to a volume of respectively. Fine particle samples from all subjects were cultured approximately 400 ml. Following ultrafiltration, the filter was washed with 200 ml of DPBS++BSA, and the wash solution was for infectious virus on MDCK cells. Confluent cells in 24-well plates (Corning, NY, USA)were inoculated with 0.1 ml of the combined with the retentate. Samples were stored at 280uC. concentrated sample diluted 1:1 in OptiMEMH I medium RNA extraction in Trizol-chloroform, reverse transcription, and (Invitrogen, Carlsbad, California). The plates were incubated at quantitative PCR were performed as previously described [1,32]. 37uC for 1 h with rocking every 15 min, and 0.8 ml of Quantitative PCR was performed using an Applied Biosystems Prism 7300 detection system (Foster City, CA) for coarse fraction OptiMEMH I media with 1 mg/ml of TPCK-trypsin was added to each well and incubated for 72–96 h. The cells were checked samples or a LightCycler 480 (Roche, Indianapolis, IN) for the fine particle fraction. Duplicate samples were analyzed using influenza daily for cytopathic effect (CPE) and if none was detected, two blind passages were performed using cell supernatant. At each A and B primers described by van Elden et al. [33] A standard curve was constructed in each assay with cDNA extracted from a passage, supernatants were tested for influenza virus by hemag- stock of influenza A (A/Puerto Rico/8/1934, Advanced Biotech- glutination (HA) assay using 0.5% chicken red blood cells. Positive nologies Incorporated, Columbia, MD) with a concentration of samples were confirmed by Flu DETECT (Synbiotics, CA, USA) PLOS Pathogens | www.plospathogens.org 6 March 2013 | Volume 9 | Issue 3 | e1003205 Influenza Virus Aerosols in Human Exhaled Breath strip test and by amplification of the hemagglutination (HA) gene Supporting Information by RT-PCR followed by sequencing. Table S1 Copy number and influenza type in five assayed samples per subject. Statistical analysis (DOCX) We analyzed the effect of surgical masks as a) log relative risk for production of any virus aerosols assuming a binomial distribution Acknowledgments using generalized estimating equations with exchangeable within- subject correlation to account for repeated measures, and b) the We wish to acknowledge the extensive laboratory contributions of Priyanka geometric mean counts of virus particles detected in exhaled Chintha without whom this would not have been completed; Matthew breath by qPCR and fractional reduction in copy number using Angel and his mentor, Daniel R. Perez, Ph.D., who provided viral culture Tobit regression analysis on log copy number with a random effect expertise, facilities, and cells. We also thank Ashook Chockalingam, Sara to account for variability between individuals. Tobit analysis was Schloth, Kesava Kalluri, Benjamin Kozak, and Linda Haggerty for their time and energy devoted to this project and Richard Martinello for helpful also used to compare coarse and fine particle fractions. Tobit comments on early drafts of the manuscript. regression avoids bias that would arise from assigning samples below the limit of detection a specific value such as zero or the Author Contributions limit divided by the square root of 2. Surgical mask use was the dependent variable. We also computed McNemar’s test for paired Conceived and designed the experiments: DKM MPF JJM. Performed the samples to examine mask effect and Spearman’s correlation experiments: DKM MPF JJM. Analyzed the data: DKM. Wrote the paper: coefficient to examine the relationship between the load in the DKM MPF BJC MLG JJM. Performed confirmatory experiments: MLG. nasopharyngeal swab and aerosol fractions. 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Langmuir AD (1980) Changing concepts of airborne infection of acute Infections: Current Research Issues Institute of Medicine (2011) Preventing contagious diseases: a reconsideration of classic epidemiologic theories. Transmission of Pandemic Influenza and Other Viral Respiratory Diseases: Ann N Y Acad Sci 353: 35–44. Personal Protective Equipment for Healthcare Workers Update 2010; Larson 26. Fabian P, McDevitt JJ, Houseman EA, Milton DK (2009) Airborne influenza EL, Liverman CT, editors: The National Academies Press. virus detection with four aerosol samplers using molecular and infectivity assays: 10. Centers for Disease Control and Prevention (2011) RFA IP-11-001 Virologic considerations for a new infectious virus aerosol sampler. Indoor Air 19: 433– evaluation of the modes of influenza virus transmission among humans. http:// grants.gov. Accessed 14 October 2012. 27. McDevitt JJ, Koutrakis P, Ferguson ST, Wolfson JM, Fabian MP, et al. (2013) 11. Killingley B, Enstone JE, Greatorex J, Gilbert AS, Lambkin-Williams R, et al. Development and performance evaluation of an exhaled-breath bioaerosol (2012) Use of a human influenza challenge model to assess person-to-person collector for influenza virus. Aerosol Sci Tech DOI 10.1080/ 02786826.2012.762973. transmission: proof-of-concept study. J Infect Dis 205: 35–43. 28. Killingley B, Enstone J, Booy R, Hayward A, Oxford J, et al. (2011) Potential 12. Alford RH, Kasel JA, Gerone PJ, Knight V (1966) Human influenza resulting role of human challenge studies for investigation of influenza transmission. from aerosol inhalation. Proc Soc Exp Biol Med 122: 800–804. Lancet Infect Dis 11: 879–886. 13. Levy BS, Wegman DH, Barron SL, Sokas RK (2005) Occupational health : 29. Carrat F, Vergu E, Ferguson NM, Lemaitre M, Cauchemez S, et al. (2008) recognizing and preventing work-related disease and injury. Philadelphia: Time lines of infection and disease in human influenza: a review of volunteer Lippincott Williams & Wilkins. 847 p. challenge studies. Am J Epidemiol 167: 775–785. 14. Centers for Disease Control and Prevention (2009) Interim Guidance for Novel 30. Roberts MG, Nishiura H (2011) Early Estimation of the Reproduction Number H1N1 Flu (Swine Flu): Taking Care of a Sick Person in Your Home. Centers for in the Presence of Imported Cases: Pandemic Influenza H1N1-2009 in New Disease Control and Prevention. http://www.cdc.gov/h1n1flu/guidance_ Zealand. Plos One 6. homecare.htm Accessed 11 February 2010. 31. Lloyd-Smith JO, Schreiber SJ, Kopp PE, Getz WM (2005) Superspreading and 15. Centers for Disease Control and Prevention (2009) Interim Recommendations the effect of individual variation on disease emergence. Nature 438: 355–359. for Facemask and Respirator Use to Reduce Novel Influenza A (H1N1) Virus 32. Fabian P, McDevitt JJ, Lee WM, Houseman EA, Milton DK (2009) An Transmission. http://www.cdc.gov/h1n1flu/masks.htm Accessed 11 February optimized method to detect influenza virus and human rhinovirus from exhaled breath and the airborne environment. J Environ Monit 11: 314–317. 16. Johnson DF, Druce JD, Birch C, Grayson ML (2009) A quantitative assessment 33. van Elden LJ, Nijhuis M, Schipper P, Schuurman R, van Loon AM (2001) of the efficacy of surgical and N95 masks to filter influenza virus in patients with Simultaneous detection of influenza viruses A and B using real-time quantitative acute influenza infection. Clin Infect Dis 49: 275–277. PCR. J Clin Microbiol 39: 196–200. PLOS Pathogens | www.plospathogens.org 7 March 2013 | Volume 9 | Issue 3 | e1003205 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS Pathogens Public Library of Science (PLoS) Journal

Influenza Virus Aerosols in Human Exhaled Breath: Particle Size, Culturability, and Effect of Surgical Masks

PLoS Pathogens , Volume 9 (3): e1003205 – Mar 7, 2013

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Public Library of Science (PLoS) Journal
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Copyright: © 2013 Milton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by the Centers for Disease Control and Prevention cooperative agreements 1U01CI000446 and 1U01IP000497, NIH grant RC1AI086900, and the U.S. Federal Aviation Administration (FAA) Office of Aerospace Medicine through the Air Transportation Center of Excellence for Airliner Cabin Environment Research (Cooperative Agreement 04-C-ACE-HU and 07-C-RITE-HU) via a subcontract from Auburn University (#06-ACER-207814). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the funding agency. Competing interests: BJC has received research funding from MedImmune Inc., and consults for Crucell MV. The authors declare that no other competing interests exist.This does not alter our adherence to all PLoS Pathogens policies on sharing data and materials.
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Research Article; Chemistry; Physical chemistry; Mixtures; Aerosols; Medicine; Infectious diseases; Viral diseases; Influenza; Chemistry; Infectious Diseases; Public Health and Epidemiology
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1553-7366
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1553-7374
DOI
10.1371/journal.ppat.1003205
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Abstract

The CDC recommends that healthcare settings provide influenza patients with facemasks as a means of reducing transmission to staff and other patients, and a recent report suggested that surgical masks can capture influenza virus in large droplet spray. However, there is minimal data on influenza virus aerosol shedding, the infectiousness of exhaled aerosols, and none on the impact of facemasks on viral aerosol shedding from patients with seasonal influenza. We collected samples of exhaled particles (one with and one without a facemask) in two size fractions (‘‘coarse’’.5 mm, ‘‘fine’’#5 mm) from 37 volunteers within 5 days of seasonal influenza onset, measured viral copy number using quantitative RT-PCR, and tested the fine-particle fraction for culturable virus. Fine particles contained 8.8 (95% CI 4.1 to 19) fold more viral copies than did coarse particles. Surgical masks reduced viral copy numbers in the fine fraction by 2.8 fold (95% CI 1.5 to 5.2) and in the coarse fraction by 25 fold (95% CI 3.5 to 180). Overall, masks produced a 3.4 fold (95% CI 1.8 to 6.3) reduction in viral aerosol shedding. Correlations between nasopharyngeal swab and the aerosol fraction copy numbers were weak (r = 0.17, coarse; r = 0.29, fine fraction). Copy numbers in exhaled breath declined rapidly with day after onset of illness. Two subjects with the highest copy numbers gave culture positive fine particle samples. Surgical masks worn by patients reduce aerosols shedding of virus. The abundance of viral copies in fine particle aerosols and evidence for their infectiousness suggests an important role in seasonal influenza transmission. Monitoring exhaled virus aerosols will be important for validation of experimental transmission studies in humans. Citation: Milton DK, Fabian MP, Cowling BJ, Grantham ML, McDevitt JJ (2013) Influenza Virus Aerosols in Human Exhaled Breath: Particle Size, Culturability, and Effect of Surgical Masks. PLoS Pathog 9(3): e1003205. doi:10.1371/journal.ppat.1003205 Editor: Ron A. M. Fouchier, Erasmus Medical Center, Netherlands Received October 21, 2012; Accepted January 9, 2013; Published March 7, 2013 Copyright:  2013 Milton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by the Centers for Disease Control and Prevention cooperative agreements 1U01CI000446 and 1U01IP000497, NIH grant RC1AI086900, and the U.S. Federal Aviation Administration (FAA) Office of Aerospace Medicine through the Air Transportation Center of Excellence for Airliner Cabin Environment Research (Cooperative Agreement 04-C-ACE-HU and 07-C-RITE-HU) via a subcontract from Auburn University (#06-ACER-207814). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the funding agency. Competing Interests: BJC has received research funding from MedImmune Inc., and consults for Crucell MV. The authors declare that no other competing interests exist. This does not alter our adherence to all PLoS Pathogens policies on sharing data and materials. * E-mail: dmilton@umd.edu . These authors contributed equally to this work. possible routes of infection, including use of fit-tested N95 Introduction respirators [3]. A year after the 2009 pandemic, there was no Transmission of influenza virus between humans may occur by greater clarity on the importance of the various modes of three routes: (1) direct or indirect contact between an infected and transmission [9]. a susceptible person, usually resulting in contamination of a The U.S. Centers for Disease Control and Prevention recently susceptible person’s hands followed by hand to respiratory mucosa funded an experimental study of person-to-person transmission to contact; (2) large droplet spray wherein droplets of respiratory fluid address this important knowledge gap [10]. However, an greater than approximately 100 mm in diameter are expelled with experimental study using intranasal inoculation to infect experi- sufficient momentum to deliver a direct hit on the respiratory mental donors [11] will need to show that the donors and naturally mucosa; and (3) aerosols generated by release of smaller, virus- infected persons shed similar virus aerosols with regard to containing droplets, as may occur during tidal breathing and quantity, particle size distribution, and infectiousness, given that coughing [1,2], that rapidly evaporate into residual particles earlier experiments suggested that intranasal inoculation requires (droplet nuclei),which are inhaled and deposited in the respiratory quantitatively larger doses and produces qualitatively milder illness tract [3–6]. There is significant evidence for each of these routes than does inoculation via aerosol [12]. [7,8], but their relative importance is not known [3]. As a result, In an occupational hygiene context, personal protection is the Institute of Medicine recommended that healthcare workers in usually the last resort, after source mitigation and environmental contact with 2009-H1N1 patients use protection against all of the controls are exhausted [13]. Thus, it is worthwhile considering PLOS Pathogens | www.plospathogens.org 1 March 2013 | Volume 9 | Issue 3 | e1003205 Influenza Virus Aerosols in Human Exhaled Breath Author Summary Table 1. Participant’s sex, symptoms, temperature, and influenza virus type. The relative importance of direct and indirect contact, large droplet spray, and aerosols as modes of influenza transmission is not known but is important in devising N Percent effective interventions. Surgical facemasks worn by pa- Number with complete data 37 100 tients are recommended by the CDC as a means of reducing the spread of influenza in healthcare facilities. We Male 30 81 sought to determine the total number of viral RNA copies a On antiviral medicine 00 present in exhaled breath and cough aerosols, whether the Asthmatic 514 RNA copies in fine particle aerosols represent infectious Flu shot this season 13 virus, and whether surgical facemasks reduce the amount of virus shed into aerosols by people infected with Flu shot previous seasons 12 32 seasonal influenza viruses. We found that total viral copies a Current smoker 924 detected by molecular methods were 8.8 times more Tachypnea 13 35 numerous in fine (#5 mm) than in coarse (.5 mm) aerosol Breathing difficulty 16 43 particles and that the fine particles from cases with the highest total number of viral RNA copies contained Lymphadenopathy 18 49 infectious virus. Surgical masks reduced the overall a Feverish 19 51 number of RNA copies by 3.4 fold. These results suggest Temperature $37.8uC10 27 an important role for aerosols in transmission of influenza Type A 21 57 virus and that surgical facemasks worn by infected persons are potentially an effective means of limiting the spread of Self-reported. influenza. At time of exhaled breath measurement. doi:10.1371/journal.ppat.1003205.t001 whether surgical facemasks could be effective as a means of source copies (Figure 1) was below the limit of detection with and without th control. The CDC recommends that persons with influenza wear facemasks; the 75 percentile dropped from 37 to below the limit surgical masks when in contact with susceptible individuals of detection with use of surgical masks. Using Tobit analysis, we [14,15]. However, there is only one report studying mask impact estimated that the geometric mean coarse fraction copy number on containment of infectious large droplet spray during influenza without a facemask was 12 (95% confidence interval (CI), 4 to 37) infection [16], and no data on surgical mask impact on release of and that the effect of facemasks was to produce a statistically infectious viral aerosols. significant 25 fold reduction in the copy number (95% CI 3.5 to In the current study of patients infected with seasonal influenza, 180, p = 0.002) to ,0.5 copies per 30 min sample. we describe the number of copies of viral RNA in two aerosol size We detected viral RNA in 78% (29 of 37) of fine particle fractions, report the culturability of virus in the fine-particle samples collected from volunteers when they were wearing a mask fraction, and the effect of surgical masks. and in 92% (34 of 37) of samples collected when they were not wearing a mask. Thus, the relative risk for any virus detection with mask versus without a mask was 0.85 and borderline statistically Results significant (CI 0.72 to 1.01; McNemar’s test p = 0.06). However, We screened 89 volunteers: 33 (37%) tested positive for the reduction in copy number was statistically significant: The influenza using the rapid test (20 influenza A and 13 influenza median number of viral copies in the fine particle fraction was 250 B) and were asked to provide exhaled breath samples. Eight with masks and 560 without masks. The geometric mean copy additional volunteers with negative rapid tests who reported a number in the fine particle fraction without a facemask was 110 cough and who had a temperature of $37.8uC were also invited to (95% CI 45 to 260) and the facemasks produced a 2.8 fold participate. In total, 38 volunteers were confirmed to have reduction in copy number (95% CI 1.5 to 5.2, p = 0.001). influenza virus infection by PCR of nasopharyngeal specimens. Combining the coarse and fine fractions, we detected viral RNA Exhaled breath data with and without a surgical mask are in 29 (78%) subjects when wearing facemasks and 35 (95%) when complete for 37 of the 38 volunteers (21 influenza A, 16 influenza not wearing facemasks (McNemar’s test p = 0.01). Surgical masks B); data for one volunteer has been excluded due to laboratory produced a 3.4 (95% CI 1.8 to 6.3) fold reduction in viral copies in error in sample processing. One of the infected subjects reported exhaled breath. receiving influenza vaccine for the current year. None of the Fine fraction copy numbers were on average 8.8 (95% CI 4.1 to subjects sneezed during the sample collection. Table 1 shows the 19) times larger than coarse fraction copy numbers. The coarse sex, symptom and fever prevalence, and influenza virus type and and fine fraction copy numbers were correlated (r = 0.60, Table 2 shows descriptive statistics for age and viral RNA copy p,0.0001). The viral load in the nasopharyngeal swab specimen, number in swabs and exhaled aerosol fractions of the 37 however, was not correlated with that in the coarse fraction volunteers with confirmed influenza infection. The viral copy (r = 0.17, p = 0.31) and only weakly with that in the fine fraction numbers in each of the five specimens for all 37 cases are shown in (r = 0.29, p = 0.08). There was no significant difference in copy Table S1. number between influenza A and B virus infection in either the We detected influenza virus RNA in the coarse fraction coarse (p = 0.28) or fine (p = 0.26) fraction. Reported asthma (particles greater than 5 mm) collected from 11% (4 of 37 (p = 0.029) and feverishness (p = 0.014) were associated with volunteers) while wearing surgical masks and from 43% (16 of significantly lower fine fraction copy numbers. However, coarse 37) while not wearing a mask (relative risk for virus detection with fraction copy numbers were not significantly impacted and mask = 0.25, 95% confidence interval (CI) 0.09 to 0.67; McNe- temperature measured at the time of testing was not associated mar’s test p = 0.003). The median number of coarse fraction viral with exhaled copy numbers. Vaccination in any prior year was PLOS Pathogens | www.plospathogens.org 2 March 2013 | Volume 9 | Issue 3 | e1003205 Influenza Virus Aerosols in Human Exhaled Breath Table 2. Descriptive statistics. Percentiles th th Min 25 Median 75 Max Age 18 18 19 20 54 Days since onset 01 235 3 4 5 6 7 Nasopharyngeal swab copy number 1.7610 8.3610 4.2610 1.8610 3.4610 Coarse particle copy number with mask 0 0 0 0 7.7610 1 4 Coarse particle copy number no mask 0 0 0 3.7610 2.9610 1 2 4 Fine particle copy number with mask 0 5 2.2610 2.5610 2.4610 1 2 2 5 Fine particle copy number no mask 0 1.1610 1.1610 5.6610 1.3610 At time of exhaled breath measurement. doi:10.1371/journal.ppat.1003205.t002 associated with a non-significant trend toward lower copy numbers report by Johnson et al [16], who detected influenza virus RNA in in coarse (p = 0.11) and fine fractions (p = 0.15); there were too few cough generated large droplet spray from 100% of influenza having received the current season’s vaccine to analyze. Self- patients over two brief sampling trials, and from 78% on each trial. reported tachypnea, breathing difficulty, smoking, and lymphade- These discrepant findings are likely due to the very different nopathy were not associated with significant shifts in exhaled copy collection techniques and particle sizes collected in these two studies. We used a specially designed aerosol sampler to collect numbers. We recovered infectious virus from fine particle samples (with particles from 0.05 to 50 mm in diameter. Johnson et al, by contrast, used simple deposition on petri dishes, and based on and without mask) produced by the two subjects with the highest particle settling rates and collection times, that method would have numbers of viral RNA copies in the fine particle fraction after blind passage on MDCK cells. Sequence analysis showed that the been unlikely to collect particles with diameters of less than approximately 50 mm because smaller particles would have two isolates were seasonal H1N1, with sequence differences from remained suspended in air and flowed around the petri dishes. each other and unrelated to any viruses present in the Veterinary Medicine laboratories at the time these samples were cultured. We view results from Johnson et al and the present study as complementary. Together the studies show that surgical masks Virus copy number (Table 3) declined with time since onset of can limit the emission of large droplet spray and aerosol droplets symptoms. In the coarse fraction, each additional day after onset larger than 5 mm [16]. However, surgical masks are not as was associated with a 6.0 fold drop in the number of virus copies efficient at preventing release of very small particles. It is well detected (95% CI 1.7 to 21 fold). Fine particles also declined with known that surgical masks are not effective for preventing time, each additional day after onset was associated with a 2.4 fold exposure to fine particles when worn as personal protection [18]. drop in the number of copies detected (95% CI 1.1 to 5.1 fold). We had hypothesized that when used as source control, exhaled droplets might be large enough prior to evaporation to be Discussion effectively captured, primarily through impaction. This appears We measured exhaled influenza viral particle copy number by to be true for virus carried in coarse particles. But the majority of quantitative RT- PCR in two particle size fractions, $5 mm virus in the exhaled aerosol appear to be in the fine fraction that (coarse) and ,5 mm (fine), and assayed the fine fraction for is not well contained. Nevertheless, the overall 3.4 fold reduction culturable virus. We observed that viral copy numbers were in aerosol copy numbers we observed combined with a nearly greater in the fine than in the coarse fraction, and recovered complete elimination of large droplet spray demonstrated by infectious virus from the fine particle fraction collected from the Johnson et al. suggests that surgical masks worn by infected two samples with the highest RNA copy numbers. These results, persons could have a clinically significant impact on transmission. combined with older data suggesting that the infectious dose via For example if one hypothesized that all transmission were due to aerosol is about two orders of magnitude lower than via large aerosol particles ,50 mm, and estimated a reproductive number droplets [12], suggest an important role for aerosols in seasonal of 1.5 for influenza (i.e. each infection generates 1.5 new influenza transmission. infections on average at the start of the epidemic) [19], then the Surgical masks nearly eliminated viral RNA detection in the use of surgical masks by every infected case could reduce the coarse aerosol fraction with a 25 fold reduction in the number of reproductive number below 1 [20]. Compliance, however, would viral copies, a statistically significant 2.8 fold reduction in copies be a major limitation resulting in lower efficacy in real-world detected in the fine aerosol fraction, and an overall statistically practice [21,22]. significant 3.4 fold reduction of viral copy number in the exhaled While it is generally assumed that large droplets shed from the aerosols. This finding supports current Centers for Disease Control respiratory tract contain infectious virus, there are limited data and Prevention recommendations that healthcare facilities en- that indicate that fine particle aerosols released from the human courage patients with influenza-like illness to don surgical respiratory tract contain infectious virus. In one previous study by facemasks as one component of an influenza infection control Lindsley et al, infectious virus was detected in 2 of 21 cough program [17]. aerosol samples, once with a sampler that did not discriminate When volunteers were not wearing surgical masks, we detected between coarse and fine particles and once in the coarse particle virus RNA in coarse particles exhaled by 43% and in fine particles fraction of a second instrument [23]. This observation, along with exhaled by 92% of influenza patients. This is in contrast to the our observation that it was possible to recover culturable virus PLOS Pathogens | www.plospathogens.org 3 March 2013 | Volume 9 | Issue 3 | e1003205 Influenza Virus Aerosols in Human Exhaled Breath Figure 1. Influenza virus copy number in aerosol particles exhaled by patients with and without wearing of an ear-loop surgical mask. Counts below the limit of detection are represented as 0.5 on the log scale. doi:10.1371/journal.ppat.1003205.g001 from the fine-particle fraction using our device demonstrates that viral RNA) represents technical difficulties in sampling and humans generate infectious influenza aerosols in both coarse and culturing exhaled breath samples or whether the vast majority of fine particle fractions. This lends support to the hypothesis that the virus exhaled by influenza A patients is actually non-infectious. aerosols may be a common pathway for influenza transmission These findings are consistent with those by Lindsley et al. [23] We among humans [8,24]. However, a clear test of the hypothesis designed the sampler specifically to overcome problems with requires intervention studies that can interrupt only one mode of existing bioaerosol samplers, including efficiently collecting sub- transmission without interfering with others [25]. micron particles into a liquid and use of appropriate buffer to preserve infectiousness [27]. We have previously shown that We only detected infectious virus in exhaled breath samples 4 5 with high (10 to 10 ) copy numbers by quantitative RT-PCR. collection on solid, dry collection media resulted in large losses of culturability [26]. Therefore, we did not attempt to culture the This implies that the ratio of total viral particles to infectious virus 3 4 2 3 was about 10 to 10 , compared with 10 to 10 for laboratory coarse fraction collected on a Teflon substrate. Subsequent studies stocks and experimental aerosols [26]. It is not yet known whether in our laboratory indicated that about 50% of the infectious virus the low recovery of infectious virus (despite high copy numbers of is lost during the concentration step of our procedure (data not PLOS Pathogens | www.plospathogens.org 4 March 2013 | Volume 9 | Issue 3 | e1003205 Influenza Virus Aerosols in Human Exhaled Breath Table 3. Copy number coarse and fine exhaled particles without surgical mask by day since onset of influenza symptoms. Number of Virus Particles Days Since Onset Particle Size Number of Cases Min Median Maximum 4 6 7 1Swab 10 2.1610 1.1610 3.4610 1 4 Coarse ,LD 2.3610 2.9610 2 5 Fine 4 6.1610 1.3610 4 5 6 2Swab 15 1.7610 1.0610 3.4610 Coarse ,LD ,LD 4.7610 1 4 Fine ,LD 2.1610 3.9610 4 6 7 3 Swab 7 2.3610 1.4610 1.0610 Coarse ,LD ,LD 1.1610 1 2 Fine 2 3.7610 5.3610 4 5 6 4 Swab 3 8.1610 4.2610 1.5610 Coarse ,LD ,LD ,LD 1 1 2 Fine 3.2610 7.5610 4.4610 Because there were only single cases studied on day 0 (day of onset) and on day 5 since onset of symptoms, only data for cases studied on days 1 through 4 after onset of symptoms are shown. doi:10.1371/journal.ppat.1003205.t003 shown), suggesting that this is one contributing factor in the low significant inter-individual variation and modeling suggests that rate of recovery of infectious virus in this study. cases with higher viral loads are disproportionately important in the spread of influenza [30,31]. Additional studies are also needed The lack of strong correlation between the viral load in the nasopharyngeal and aerosol samples is possibly of interest. This to determine how aerosol generation correlates with symptoms may merely be a result of nasopharyngeal sample variability; in (including milder disease), presence of other health conditions, age (we studied a narrow age distribution), and co-infection with other future studies, control for sample quality by PCR of a cellular gene may be helpful. Our sampler, as is the case with all samplers for respiratory viruses so that recommendations for infection control fine and ultrafine particles, has an upper limit to the size droplet can be critically evaluated. that can be pulled into its inlet airstream. Thus, a second possible explanation for the lack of correlation is that the nasopharynx is Methods primarily a source for very large droplets (.50 mm) that we would Patient population not have detected. Furthermore, none of our subjects sneezed; an We recruited volunteers with influenza-like illness from the Lowell, efficient method of generating droplets from the upper respiratory MA community, primarily among students and staff of the University tract. This may imply that the smaller droplets we detected were of Massachusetts, beginning January 29 and ending March 12, 2009. generated in the lower respiratory tract and that the viral load at The study protocol was approved by the Institutional Review Boards that location is not strongly correlated with the nasopharyngeal of the University of Massachusetts Lowell, Lowell General Hospital, load. Alternatively, shedding into aerosol droplets may be driven and Saints Memorial Hospital, Lowell, MA. Oral informed consent by other host factors (e.g. asthma, symptom severity, and immune was obtained by providing each subject with a detailed consent response), co-infection with other agents, virus factors affecting information form. Collection of a signed copy of the form was waived release from the epithelium, or the nature of the resident because it would have been the only personally identifiable microbiome. If shedding into aerosol is determined in large part information retained by this minimal risk study. by the location of infection in the respiratory tract, this may have Volunteers learned of the study through flyers and notices implications for experimental studies of transmission [11,28]. Such posted on campus and by referral from health care providers. We studies will need to monitor aerosol shedding to determine screened self-referred volunteers by telephone for influenza-like whether nasal inoculation of donors results in aerosol shedding illness (ILI). Persons who reported onset of fever and cough within that mimics naturally acquired infection to validate the experi- the preceding 72 hours or were referred by a health care provider mental design and aid the interpretation of results. were invited to the laboratory for testing. We collected a Most of the viral aerosol generation we observed occurred nasopharyngeal specimen using a flocked swab (501CS01, Copan during the first days of symptomatic illness (Table 3), consistent Diagnostics, Murrieta, CA) and temperature was taken with a with studies of shedding monitored by nasal washes [29]. We digital ear thermometer (Model 18-200-000, Mabis Healthcare, studied each individual on only one occasion and, by design, have Waukegan, IL). All volunteers with a temperature $37.8uC and a little data beyond day 3. Further longitudinal studies of viral cough and volunteers without fever who provided a nasopharyn- aerosol generation are needed to confirm these findings. New geal specimen positive for influenza by point of care testing study designs will be needed to examine aerosol generation before (QuikVue Influenza A/B, Quidel Corp., San Diego, CA) were and on the day of symptom onset in community acquired invited to provide exhaled breath samples, answer a questionnaire, infection. A limitation of our study is that we recruited patients and provide a second nasopharyngeal specimen for analysis by with certain signs and symptoms or who were positive on a rapid PCR. Only subjects with influenza infection confirmed by PCR test or had fever, and therefore our data could be biased towards were included in the data analysis. patients with higher viral loads [21]. However, we still observed PLOS Pathogens | www.plospathogens.org 5 March 2013 | Volume 9 | Issue 3 | e1003205 Influenza Virus Aerosols in Human Exhaled Breath Exhaled breath collection We collected exhaled breath with the subject seated in front of the inlet for a sampler designed for human exhaled breath collection, Figure 2, (G-II) described in detail by McDevitt et al. [27] Briefly, the G-II inlet was cone shaped so that the subject’s face was situated inside the large end of an open cone with air drawn continuously around the subject and into the sampler. The cone allows the subject to breathe normally and unlike use of a mouthpiece, the subject could also wear a mask. The cone served as a capture type ventilation hood allowing collection of exhaled breath with minimal fugitive emissions even when the subject was wearing a mask with resultant redirection of flow. Intake air (130 L/min) flowed through a conventional slit impactor that collected particles larger than 5 mm on a Teflon surface (‘‘coarse’’ particle fraction). To collect a ‘‘fine’’ particle fraction, water vapor was condensed on the remaining particles, which created droplets large enough to be captured by a 1.0-mmslit impactor. The 1.0-mm impactor was composed of a slit and a steel impaction surface sealed inside a large reservoir. Impacted droplets drained from the impaction surface into a buffer- containing liquid in the bottom of the reservoir. Concentrated buffer was pumped into the reservoir during collection to match the accumulation of water from collected droplets and maintain phosphate buffered saline with 0.1% bovine serum albumin throughout collection. The sampler was shown to be 85% efficient for particles greater than 50 nm in diameter and was comparable to the SKC BioSampler for detection and recovery of influenza A/PR/8/34 H1N1 by PCR and culture. Between subjects, the apparatus was disassembled and cleaned with a 0.5% hypochlorite solution. Exhaled particles were collected for 30 minutes while the subject wore an ear-loop surgical mask (Kimberly-Clark, Roswell, GA) and then for 30 minutes without a mask. Subjects were asked to cough 10 times at approximately 10-minute intervals for a total of 30 coughs during each 30 minute sample. One subject coughed frequently such that forced coughs were not required. No subjects Figure 2. Exhaled breath collection system. Each volunteer sat as shown with face inside the inlet cone of the human exhaled breath air were observed to sneeze. sampler inside a booth supplied with HEPA filtered, humidified air for 30 min while wearing an ear-loop surgical mask. Three times during the Sample analysis 30 min each subject was asked to cough 10 times. After investigators Immediately after collection, the Teflon impaction surface was changed the collection media, the volunteer sat in the cone again, without wearing a surgical mask, for another 30 min with coughing as removed and temporarily stored at 220uC. The impactors were before. scraped with a flocked swab wetted with Dulbecco’s phosphate doi:10.1371/journal.ppat.1003205.g002 buffered saline with calcium and magnesium (Hyclone, Thermo Scientific, Waltham, MA) with 0.1% bovine serum albumin 3.0610 virus particles per mL or a stock of influenza B (B/Lee/ (DPBS++BSA). The swab was eluted in 600 ml of DPBS++BSA for 1940, Advanced Biotechnologies Incorporated, Columbia, MD) 1 minute with vortexing. The resulting sample was stored at with a concentration of 8.6610 virus particles per mL as 280uC. determined by electron microscopy. Results are expressed as the The fine particle fraction collected in DPBS++BSA buffer (100 total number of virus particles by reference to the standard curve, to 150 ml volume) was maintained at 4uC and concentrated by rounded to the closest integer value. The limits of detection were 6 ultrafiltration using Amicon Ultra 15 filter units with a molecular and 11 viral RNA copies per qPCR well for influenza A and B weight cut off of 100 kD (Millipore, Bedford, MA) to a volume of respectively. Fine particle samples from all subjects were cultured approximately 400 ml. Following ultrafiltration, the filter was washed with 200 ml of DPBS++BSA, and the wash solution was for infectious virus on MDCK cells. Confluent cells in 24-well plates (Corning, NY, USA)were inoculated with 0.1 ml of the combined with the retentate. Samples were stored at 280uC. concentrated sample diluted 1:1 in OptiMEMH I medium RNA extraction in Trizol-chloroform, reverse transcription, and (Invitrogen, Carlsbad, California). The plates were incubated at quantitative PCR were performed as previously described [1,32]. 37uC for 1 h with rocking every 15 min, and 0.8 ml of Quantitative PCR was performed using an Applied Biosystems Prism 7300 detection system (Foster City, CA) for coarse fraction OptiMEMH I media with 1 mg/ml of TPCK-trypsin was added to each well and incubated for 72–96 h. The cells were checked samples or a LightCycler 480 (Roche, Indianapolis, IN) for the fine particle fraction. Duplicate samples were analyzed using influenza daily for cytopathic effect (CPE) and if none was detected, two blind passages were performed using cell supernatant. At each A and B primers described by van Elden et al. [33] A standard curve was constructed in each assay with cDNA extracted from a passage, supernatants were tested for influenza virus by hemag- stock of influenza A (A/Puerto Rico/8/1934, Advanced Biotech- glutination (HA) assay using 0.5% chicken red blood cells. Positive nologies Incorporated, Columbia, MD) with a concentration of samples were confirmed by Flu DETECT (Synbiotics, CA, USA) PLOS Pathogens | www.plospathogens.org 6 March 2013 | Volume 9 | Issue 3 | e1003205 Influenza Virus Aerosols in Human Exhaled Breath strip test and by amplification of the hemagglutination (HA) gene Supporting Information by RT-PCR followed by sequencing. Table S1 Copy number and influenza type in five assayed samples per subject. Statistical analysis (DOCX) We analyzed the effect of surgical masks as a) log relative risk for production of any virus aerosols assuming a binomial distribution Acknowledgments using generalized estimating equations with exchangeable within- subject correlation to account for repeated measures, and b) the We wish to acknowledge the extensive laboratory contributions of Priyanka geometric mean counts of virus particles detected in exhaled Chintha without whom this would not have been completed; Matthew breath by qPCR and fractional reduction in copy number using Angel and his mentor, Daniel R. Perez, Ph.D., who provided viral culture Tobit regression analysis on log copy number with a random effect expertise, facilities, and cells. We also thank Ashook Chockalingam, Sara to account for variability between individuals. Tobit analysis was Schloth, Kesava Kalluri, Benjamin Kozak, and Linda Haggerty for their time and energy devoted to this project and Richard Martinello for helpful also used to compare coarse and fine particle fractions. Tobit comments on early drafts of the manuscript. regression avoids bias that would arise from assigning samples below the limit of detection a specific value such as zero or the Author Contributions limit divided by the square root of 2. Surgical mask use was the dependent variable. We also computed McNemar’s test for paired Conceived and designed the experiments: DKM MPF JJM. Performed the samples to examine mask effect and Spearman’s correlation experiments: DKM MPF JJM. Analyzed the data: DKM. Wrote the paper: coefficient to examine the relationship between the load in the DKM MPF BJC MLG JJM. Performed confirmatory experiments: MLG. nasopharyngeal swab and aerosol fractions. 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Radonovich LJ, Jr., Cheng J, Shenal BV, Hodgson M, Bender BS (2009) spread of infection? Indoor Air 16: 335–347. Respirator tolerance in health care workers. JAMA 301: 36–38. 7. Brankston G, Gitterman L, Hirji Z, Lemieux C, Gardam M (2007) Transmission 23. Lindsley WG, Blachere FM, Thewlis RE, Vishnu A, Davis KA, et al. (2010) of influenza A in human beings. Lancet Infect Dis 7: 257–265. Measurements of airborne influenza virus in aerosol particles from human 8. Tellier R (2006) Review of aerosol transmission of influenza A virus. Emerg coughs. PLoS One 5: e15100. Infect Dis. pp. 1657–1662. 24. Tellier R (2009) Aerosol transmission of influenza A virus: a review of new 9. Committee on Personal Protective Equipment for Healthcare Personnel to studies. J R Soc Interface 6 Suppl 6: S783–790. Prevent Transmission of Pandemic Influenza and Other Viral Respiratory 25. Langmuir AD (1980) Changing concepts of airborne infection of acute Infections: Current Research Issues Institute of Medicine (2011) Preventing contagious diseases: a reconsideration of classic epidemiologic theories. Transmission of Pandemic Influenza and Other Viral Respiratory Diseases: Ann N Y Acad Sci 353: 35–44. Personal Protective Equipment for Healthcare Workers Update 2010; Larson 26. Fabian P, McDevitt JJ, Houseman EA, Milton DK (2009) Airborne influenza EL, Liverman CT, editors: The National Academies Press. virus detection with four aerosol samplers using molecular and infectivity assays: 10. Centers for Disease Control and Prevention (2011) RFA IP-11-001 Virologic considerations for a new infectious virus aerosol sampler. Indoor Air 19: 433– evaluation of the modes of influenza virus transmission among humans. http:// grants.gov. Accessed 14 October 2012. 27. McDevitt JJ, Koutrakis P, Ferguson ST, Wolfson JM, Fabian MP, et al. (2013) 11. 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Philadelphia: Time lines of infection and disease in human influenza: a review of volunteer Lippincott Williams & Wilkins. 847 p. challenge studies. Am J Epidemiol 167: 775–785. 14. Centers for Disease Control and Prevention (2009) Interim Guidance for Novel 30. Roberts MG, Nishiura H (2011) Early Estimation of the Reproduction Number H1N1 Flu (Swine Flu): Taking Care of a Sick Person in Your Home. Centers for in the Presence of Imported Cases: Pandemic Influenza H1N1-2009 in New Disease Control and Prevention. http://www.cdc.gov/h1n1flu/guidance_ Zealand. Plos One 6. homecare.htm Accessed 11 February 2010. 31. Lloyd-Smith JO, Schreiber SJ, Kopp PE, Getz WM (2005) Superspreading and 15. Centers for Disease Control and Prevention (2009) Interim Recommendations the effect of individual variation on disease emergence. Nature 438: 355–359. for Facemask and Respirator Use to Reduce Novel Influenza A (H1N1) Virus 32. Fabian P, McDevitt JJ, Lee WM, Houseman EA, Milton DK (2009) An Transmission. http://www.cdc.gov/h1n1flu/masks.htm Accessed 11 February optimized method to detect influenza virus and human rhinovirus from exhaled breath and the airborne environment. J Environ Monit 11: 314–317. 16. Johnson DF, Druce JD, Birch C, Grayson ML (2009) A quantitative assessment 33. van Elden LJ, Nijhuis M, Schipper P, Schuurman R, van Loon AM (2001) of the efficacy of surgical and N95 masks to filter influenza virus in patients with Simultaneous detection of influenza viruses A and B using real-time quantitative acute influenza infection. Clin Infect Dis 49: 275–277. PCR. J Clin Microbiol 39: 196–200. PLOS Pathogens | www.plospathogens.org 7 March 2013 | Volume 9 | Issue 3 | e1003205

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