Rationale: Respiratory syncytial virus (RSV) is a highly contagious pathogen with a huge global health impact. It is a major cause of hospital-acquired infection; a large number of those exposed develop infection. Those infected in hospital are at increased risk of a severe clinical course. Prevention of nosocomial spread currently focuses on spread by hand and large droplets. There is little research evidence to determine if aerosol spread of infectious RSV is possible.
Objectives: To determine if the air surrounding infants with RSV-positive bronchiolitis contains RSV in aerosolized particles that remain capable of causing infection.
Methods: The amount of RSV contained in aerosolized particles produced by infants with bronchiolitis due to RSV was measured using viable impactor sampling. The ability of RSV contained in these particles to infect healthy and chronic obstructive pulmonary disease (COPD) human ciliated respiratory epithelium was determined.
Results: We showed for the first time that infants with RSV-positive bronchiolitis nursed in a ward setting or ventilated in intensive care produced large numbers of aerosol particles containing RSV that remained infectious and were capable of infecting healthy and COPD human ciliated epithelium. A significant amount of RSV was found in particles with aerodynamic diameters less than 5 μm.
Conclusions: Many of the aerosolized particles that contained RSV in the air surrounding infants with bronchiolitis were sufficiently small to remain airborne for a significant length of time and small enough to be inhaled and deposited throughout the respiratory tract. It is likely that this leads to spread of infection to others, with dissemination of infection throughout the respiratory tract.
Nosocomial infection with respiratory syncytial virus (RSV) is associated with the risk of a severe clinical course. It is currently believed that large droplets and hand contamination spread RSV, and most infection control strategies are based on prevention of spread by direct contact or by large droplets. Spread of RSV by aerosol is not considered important. As a consequence of this, infants with RSV bronchiolitis are frequently nursed in open bays on general wards and in the open ward area of intensive care units.
This study shows that the air surrounding both spontaneously breathing and ventilated infants with RSV-positive bronchiolitis contains large amounts of aerosolized RSV, which remains infectious and is contained in particles small enough to remain airborne for considerable lengths of time and to be inhaled into the lower respiratory airways. This study has significant implications for the current infection control strategies in many hospitals and calls into question the practice of nursing infants with RSV bronchiolitis in an open ward setting in general wards and in open areas on intensive care.
In a single year, more than 33 million new episodes of respiratory syncytial virus (RSV)-associated acute lower respiratory infection occur in children aged younger than 5 years (1). RSV is also a major cause of morbidity in adults (1, 2), especially in those with cardiopulmonary disease, frail older adults, and severely immune-compromised individuals in whom mortality rates are high (3).
RSV is highly contagious in the community and on hospital wards (4, 5), with almost all children having evidence of infection by 2 years of age (6). Up to 40% (4, 5) of hospitalized infants in contact with RSV become infected, which is of concern because the risk of a severe clinical course is significantly increased in hospital-acquired RSV infection (7). Because no drugs are available to treat those infected with RSV, infection control strategies are of particular importance.
Most infection control strategies for RSV are based on prevention of spread by contact or large droplet transmission (5, 8, 9). RSV can survive on nonporous surfaces, skin, and gloves for many hours (10, 11), and there is evidence that RSV is spread by contamination of hands. Considerable controversy still exists as to whether spread of infection may also occur by inhalation of aerosolized airborne particles containing RSV; it is acknowledged that research evidence is lacking in this area (12). The perceived lack of infection by RSV contained in aerosol particles produced by infants with bronchiolitis is largely based on a single study in which adults sitting 1.82 m from infants did not become infected (13). However, the individual susceptibility to infection may vary, and the dose of RSV needed to infect seronegative infants has been shown to be very low (10). Although there is evidence of airborne RSV in health care facilities, as measured by polymerase chain reaction (PCR), to date no study has determined if RSV aerosols produced by those infected remain infectious and how much RSV is contained in aerosolized particles that may be inhaled (9, 14).
In this study, for the first time to our knowledge, we provide evidence that large numbers of aerosolized particles containing RSV that remains infectious are found in the air surrounding infants infected with RSV who are nursed in general pediatric wards and intensive care units.
This study has significant implications for the current infection control strategies in many hospitals and calls into question the practice of nursing infants with RSV bronchiolitis in an open ward setting, particularly with other infants in whom the etiology of their respiratory problem has not been identified.
Recruitment took place over two bronchiolitis seasons at Leicester Royal Infirmary. Recruited subjects were nursed in either an open bay of six cots (“bronchiolitis bay”: Figure 1A) reserved for infants clinically suspected of having bronchiolitis, or in an isolation cubicle (“cubicle”: Figure 1B). The bronchiolitis bay was created due to the lack of isolation cubicles. Mechanically ventilated infants and young children nursed on the children’s intensive care unit (CICU) were also recruited. The CICU consists of an “open CICU bay” of five cots or beds (Figure 1C) and CICU cubicles.
The research team did not interfere with clinical management of patients. Infants suspected of having clinical bronchiolitis had nasopharyngeal aspirates performed within 24 hours of admission. We used a UK definition of bronchiolitis: “a seasonal viral illness characterized by fever, nasal discharge and dry, wheezy cough. On examination of children with bronchiolitis there are fine inspiratory crackles and/or high pitched expiratory wheeze” (15). Four older children (older than 2 yr), three nursed in cubicles on the general ward and one ventilated in the open CICU bay, who were RSV-positive were investigated to determine if they produced aerosolized particles containing infectious RSV.
A control group of infants with no respiratory problems (e.g., prolonged jaundice, feeding difficulties, orthopedic) nursed in cubicles on the pediatric ward was recruited.
The project had ethical approval from the East Midlands National Research Ethics Service Committee (08/H0408/128), and written informed consent was obtained from parents/guardians of the children.
Detection of RSV A and B was performed by PCR (7–9, 16, 17). Samples were also tested for influenza A and B, parainfluenza 1,2,3,4, and adenovirus. Virology results of all patients on the general ward and in the CICU at the time of sampling from the index case were recorded.
A viable impactor (Westech 6-stage Microbial sampler; Westech Scientific, Upper Stondon, UK) was used to collect aerosol particles and determine aerodynamic size. This system has previously been used for sampling airborne influenza virus in liquid medium (18) and for cough generated aerosols of Mycobacterium tuberculosis (19) and Pseudomonas aeruginosa (20).
The impactor sampled airborne particles at 28.3 L/min for 30 minutes. The impactor consists of six stages that allow fractionation of the particles into the following aerodynamic size ranges: ≥7 μm (stage 1); 4.7 to 7 μm (stage 2); 3.3 to 4.7 μm (stage 3); 2.1 to 3.3 μm (stage 4); 1.1 to 2.1 μm (stage 5); and 0.65 to 1.1 μm (stage 6). Particles were impacted into separate Petri dishes containing 20 ml of media (Gibco RPMI Media 1640; Life Technologies, Grand Island, NY) with added penicillin (50 μg/ml), streptomycin (50 μg/ml), and Fungizone (1 μg/ml) (Life Technologies).
Quantification of viable RSV was performed using type 2 alveolar basal epithelial cells (A549). Cells were propagated in RPMI Media 1640 (as previously described), supplemented with 10% (v/v) fetal bovine serum (Life Technologies), penicillin (50 μg/ml), streptomycin (50 μg/ml), Fungizone (1 μg/ml), and incubated at 37°C in 5% CO2. Cells were seeded into 96 well plates to form an 80% confluent monolayer. Triplicate wells were then exposed to 200 μl of a log dilution series of the impacted air sample at 37°C in 5% CO2 for 2 hours. Samples were removed using two culture medium washes and incubated for 48 hours (21).
RSV infection was detected by direct immunofluorescent staining with fluorescein isothiocyanate–conjugated monoclonal antibody (Light Diagnostics RSV DFA; EMD Millipore Corporation, Billerica, MA) directed against RSV surface glycoprotein. Detection of fluorescent cells was performed using a 40× objective fluorescent microscope. Numbers of immunofluorescent plaques per well were counted, and the total plaque-forming units (PFUs) in each sample were calculated.
The impactor was positioned at a distance of 1 m from and level with the head of the study infant (the index case). Infants in the general pediatric ward were nursed in a bay of six cots. Other infants with clinically suspected bronchiolitis were also nursed in the same bay (Figure 1A). All were tested for viral infection during their stay. Some infants and young children with RSV-positive bronchiolitis or a lower respiratory tract infection in whom RSV had been isolated were nursed in a ward cubicle (Figure 1B). In bays where more than one infant was infected with RSV, the infant most recently admitted was chosen as the index case. After initial impactor sampling, a further sample was obtained in the bronchiolitis bay with the impactor placed in the middle of the bay 5 m from five of the index cases. In the same five cases, further impactor sampling was performed 10 m from the index patient, in the vicinity of the ward nursing station. Impactor sampling was repeated 2 hours after the index case (four of the infants nursed in cubicles and four nursed in the bronchiolitis bay) had been discharged from hospital. Two hours was chosen because this was the average turnaround time before the next patient occupied the same cubicle or cot during the study period.
In intensive care, impactor sampling was performed in nine infants with RSV-positive bronchiolitis and one young child with RSV infection. Of the seven infants in the open intensive care ward (Figure 1C), six were ventilated and one was breathing spontaneously. Three infants studied were nursed in isolation cubicles. In intensive care, the impactor was positioned 1 m from and level with the head of the patient. Samples were collected 5 minutes after clinically indicated endotracheal suctioning. A further sample was obtained in the intensive care open ward area 5 m away from the index patient 10 minutes after the initial sample collection. Temperature and relative humidity of the room were documented.
|Location||Sex||Duration of Symptoms (d)||Nasal Cannula Oxygen||Weight (kg)||Age (wk)||PFU per Liter of Room Air||Total PFU/30-min Sampling Period|
|>7 μm||4.7–7.0 μm||3.3–4.7 μm||2.1–3.3 μm||1.1–2.1 μm||0.65–1.1 μm||Total|
|Location||Sex||Duration of Symptoms (d)||Weight (kg)||Age (wk)||PFU per Liter of Room Air||Total PFU/30 min of Impaction|
|>7 μm||4.7–7.0 μm||3.3–4.7 μm||2.1–3.3 μm||1.1–2.1 μm||0.65–1.1 μm||Total|
Human ciliated epithelial cells were obtained from healthy volunteers (n = 3) or COPD patients (n = 2) by brushing the inferior nasal turbinate with a 2-mm cytology brush (Keymed, Southend-on-Sea, UK). None of the subjects were taking medications, and none had a symptomatic upper respiratory tract infection in the preceding 6 weeks. Parents and/or guardians of all individuals gave their consent to be included in the study, and all samples were obtained with the parents’ and/or guardians’ permission and with ethical approval by the North West Liverpool East Research Ethics Committee (14/NW/0128). Cells were grown at an air–liquid interface to allow differentiation of the epithelial subtypes as described previously (12, 22). Two hours before infection, cell cultures were fed with fresh medium, and 200 μl of the impacted sample captured from stage 3 (three wells) and stage 4 (four wells) from two separate intensive care patients was applied to the apical surface for 1 hour at 37°C and then removed. Control wells received bronchial epithelial basal medium alone. The infection was allowed to continue for 7 days. Cells were fixed, stained, and imaged as previously described (12).
Data was collected prospectively, and results are expressed as mean ± SD and range. Statistical analysis was performed using Prism 5 (GraphPad, San Diego, CA). The significance of any difference in PFUs was determined using an unpaired t test to obtain a two-tailed P value. A one-way analysis of variance test was used when comparing more than two groups. A P value of less than 0.05 was regarded as significant.
Twenty-four patients admitted to the general pediatric ward with RSV infection were recruited as the index cases. Nine infants with RSV-positive bronchiolitis were nursed in the bronchiolitis bay. Twelve infants with RSV bronchiolitis and three older children with RSV infection who had a lower respiratory tract infection were nursed in ward cubicles. Age, sex, weight, and duration of symptoms are shown in Table 1. Another nine patients with RSV bronchiolitis and one older child with RSV infection were recruited from pediatric intensive care (Table 1). Seven were nursed in the open intensive care, six of whom were ventilated. Three infants were ventilated in isolation cubicles. A control group of infants (n = 8) who did not have respiratory problems, but were admitted to the Children's Hospital for other reasons, were recruited.
RSV A was detected in aerosolized particles collected by the impactor in all but one of the index cases (Tables 1 and 2). Two patients nursed in a cubicle in the general ward had a viral coinfection with influenza (H1N1). Table 1 shows the total amount of RSV contained in aerosolized particles captured during impactor sampling and the amount of RSV in different aerodynamic particle size ranges. The mean ± SD number of PFUs of RSV in aerosol particles for infants nursed in the bay was 315,189 ± 313,714 (range 82,600–1,120,000). A substantial amount of RSV was contained in particles less than 4.7 μm (220,011 ± 190,075; range, 61,000–680,000), a particle size for which inhalation into the lower respiratory tract is likely. The amount of RSV in aerosol particles, when sampling was performed in the cubicles of infected index cases (Table 1) (60,047 ± 93,056; range 100–313,000), was significantly less than that after sampling in the bronchiolitis bay (P = 0.04). Again, a substantial amount of RSV was contained in particles less than 4.7 μm (34,607 ± 45,093; range <100–117,000). No infectious RSV was detected after impactor collection from the eight infants from the control group, who were all nursed in cubicles on pediatric wards during the study period.
Aerosol collection 5 m away from the RSV-positive index case in the bronchiolitis bay detected infectious virus, although a significant reduction was seen (999,700 ± 98,705 at 1 m to 378,100 ± 10,7331 at 5 m) (Figure 2A). Aerosol sampling 10 m from the index case did not detect RSV (P = 0.009). Table 3 shows details of the viral status of other infants and children nursed in the bay at the time of the initial impactor sampling.
|Bed reference for the bronchiolitis bay on the general ward*|
|1||2.1 × 105||—||—||—||—||—|
|2||(Negative)||—||(MPV)||(Negative)||—||3.2 × 105|
|3||2.6 × 105||—||—||(Negative)||—||RSV|
|4||1.4 × 105||—||—||—||—||—|
|5||—||—||—||RSV||—||1.1 × 106|
|6||RSV||RSV||RSV||RSV||3.5 × 105||RSV|
|7||RSV||RSV||1.5 × 105||RSV||RSV||RSV|
|8||—||—||—||RSV||2.0 × 105||RSV|
|9||(Negative)||(Negative)||(RSV)||8.3 × 104||(RSV)||(RSV)|
|Bed reference for open CICU bay†|
|1||RSV||(Negative)||3.86 × 105||—||(Negative)|
|2||2.25 × 105||RSV||(Negative)||—||—|
|3||RSV||4.74 × 104||(Negative)||(Negative)||—|
|4||—||RSV||3.24 × 105||—||—|
|5||—||RSV||RSV||6.9 × 105||—|
|6||—||RSV||—||(Negative)||4.13 × 105|
Measurements taken 2 hours after three of the four patients studied were discharged from their cubicle showed a reduction in RSV. In the fourth case, no RSV was detected from both the impactor run where the index case was present or after discharge (Figure 3A) (27,850 ± 25,452 before discharge to 6,175 ± 7,442 2 hours after discharge). Similarly, there was a reduction in the amount of RSV detected when measurements were taken in the bronchiolitis bay after discharge of each of the index cases infected with RSV (Figure 3B) (196,650 ± 113,657 before discharge to 68,100 ± 58,978 2 hours after discharge).
Impactor samples were collected from 10 RSV-positive patients with clinical bronchiolitis, including 1 older child with an RSV infection on the CICU. Six of these children were ventilated in the open intensive care ward and three in isolation cubicles. One infant nursed in the open intensive care ward was not ventilated. The mean number of PFUs of RSV per impactor run from those ventilated in the open unit was 347,567 ± 213,868 (range, 47,400–690,000). Fewer PFUs were found in cubicles of ventilated infants infected with RSV (281,000 ± 50,408; range, 226,000–325,000). When the impactor was placed 5 m away from the index case with RSV, there was a significant reduction in infectious RSV in aerosol particles from a mean of 325,378 ± 174,156 to 88,689 ± 75,813 PFUs (P = 0.0018) per impactor collection (Figure 2B). The mean room temperature on the general ward was 22.9 ± 1.5°C, and the mean relative humidity was 36.7 ± 5.7%, and for CICU, these measurements were 22.5 ± 1°C and 29.6 ± 3.9%, respectively.
Monolayer cultures of respiratory epithelial cells infected with impacted aerosols showed that the cells were readily infected with RSV (Figure 4A). Samples taken from stages 1 to 6 of the viable impactor all showed infection of the respiratory epithelial cells (Figure 4B). Similar levels of cytopathology were observed between sample wells, indicating that virus behavior was not altered by size of the aerosol particle containing RSV.
Because ciliated epithelial cells are preferentially infected in RSV infection (12, 23), we infected ciliated primary epithelial cell cultures with the impacted samples. Human respiratory epithelial cells from a healthy donor and a patient with COPD were infected with aerosol collected in the impactor, and were fixed and stained with antibody-specific RSV proteins that are present on the surface of infected cells, and when relevant, for cilia (acetylated tubulin) (Figures 4C and 4D). Most of the infected cells that displayed positive staining for RSV antigen (green) were also positively stained for cilia (red), indicating preferential infection of ciliated cells (Figure 4C). RSV antigen was observed on the apical surface of ciliated cells and on the ciliary axoneme. No viral antigens were observed in mock-infected control wells (data not shown).
This study demonstrates, for the first time, that the room air surrounding infants and young children nursed in hospital wards and those ventilated in intensive care, who have bronchiolitis or lower respiratory tract infections caused by RSV, is laden with large numbers of aerosolized particles containing infectious RSV capable of infecting ciliated cells, the target of RSV in the respiratory tract (12, 23). A significant level of RSV was found in particles with aerodynamic diameters small enough to remain airborne for many hours and small enough to be inhaled into the lower respiratory tract. These findings suggest that transmission of RSV may occur by aerosol spread in addition to other accepted routes of transmission (10). This is important because of the very high rates of nosocomial infection and increased risk of severe clinical disease seen in those exposed to infectious virus (5, 7).
Larger particles containing RSV would be expected to drop quickly and pose a risk for transmission after deposition on surfaces. The smaller particles containing RSV are likely to remain airborne for a considerable amount of time. Pasquarella and colleagues (24) predict that a 1-μm particle descends at 10 cm/h, a 5-μm particle at 2.7 m/h, and 10-μm particles at 11 m/h. Particles less than 1 μm are likely to remain airborne for many hours, if not days. Our finding that significant amounts of RSV were contained in very small particles is consistent with the observation that infectious aerosolized particles of RSV were still present in the cubicle or bay 2 hours after the infected index cases were discharged. Our results also suggest that the infants and children studied with RSV infection were still generating aerosolized particles containing infectious RSV when discharged from hospital and are an infection risk.
In the UK, it is still common practice in many hospitals to cohort babies with a clinical presentation suggesting bronchiolitis into bays due to a lack of isolation cubicles. In this study, we confirmed cohorting was based on a clinical diagnosis of bronchiolitis rather than on a virus-specific basis, with RSV-positive babies nursed in bays with infants from whom a virus had not been isolated. Such infants, including one infected with human metapneumovirus, would have been exposed to aerosolized RSV. Because viral testing was only performed on admission, it was not possible to determine if infants with a negative result subsequently became infected with RSV.
In the bays, the amount of RSV contained in aerosolized particles was approximately five times higher than that found in the presence of RSV-positive children nursed in cubicles. The most likely explanations for this are that other infants in the bay may also be producing aerosols containing RSV or differences in air exchanges between the cubicles and the ward. However, the air exchange rate for cubicles and the general ward are both documented as 6 air exchanges per hour, making this less likely an explanation. Infants in the bronchiolitis bay were younger than those nursed in cubicles. Our study design did not allow us to determine if younger infants produced more aerosolized RSV, and further work is required to determine this.
In the busy winter season, it is common practice to ventilate children with RSV bronchiolitis in an open intensive care ward setting with infected children placed between children who do not have RSV infection. We found large amounts of RSV in aerosolized particles in the surrounding air of all of the patients with RSV infection we studied. The large amount of infectious RSV contained in particles in room air was surprising because infants were ventilated, and the air exchange rate for intensive care was 10 air exchanges per hour (25). The detection of high levels of RSV may be due in part to sampling the surrounding air 5 minutes after suctioning of the endotracheal tube. In the intensive care unit, suctioning was routinely performed in an open, rather than a closed, fashion. Although it is likely that suctioning enhanced production of the aerosol, further work is required to confirm this. The amount of aerosolized particles containing RSV was reduced when the air was sampled 5 m from the infected infant, but RSV-containing particles were found in all cases.
Aerosol particles more than 5 μm in diameter tend to impact in the nose, whereas particles less than 5 μm are likely to reach the lower airways, with very small particles of approximately 1 μm likely to reach the small airways (26). Although the production of aerosol particles may lead to primary infection of the nose and lower airways of uninfected patients, it is possible that generation of RSV-laden aerosols by the infected patient, that they then inhale, disseminates infection throughout their lower airways. For example, an infant with RSV of 9 months of age would on average breathe in 3,697 L of room air per day, which would contain significant amounts of RSV (27).
Much of the current guidance for curtailing the spread of RSV is based on a single study in which adult volunteers were in close contact with an infant with RSV or adults touched surfaces contaminated by the secretions of babies with RSV. They then rubbed their eyes and put their fingers up their nose and became infected. Adult volunteers who sat 1.82 m from an infected child did not become infected. This suggested that spread was by large droplets or self-inoculation after contact with contaminated surfaces, and transmission via the aerosol route was discounted. As a consequence, it was suggested that hand washing should be stressed and other procedures that may be helpful could include the care of contaminated surfaces and gowns (13). Based on this information (5), it may be argued that cohorting infants suspected of RSV bronchiolitis in bays, with appropriate hand-washing precautions, would be adequate to prevent transmission of RSV (5, 28).
It is believed that the dose of wild-type RSV required to infect infants for the first time is very low. When a wild-type RSV was administered intranasally to seropositive volunteers in doses of 160 to 640 50% tissue culture infective dose (TCID50), 83% were infected (29). In another trial, most seropositive children were infected with 104 TCID50 of attenuated RSV vaccines. One of the three seronegative infants tested was infected by a much lower dose of 30 to 40 TCID50 (10), suggesting that the dose of RSV required to infect an infant would be even less, and that in natural infection, the initiation of infection most probably occurs with a very small infectious dose (30). The very high infectivity rate of RSV, with almost all 2 year olds showing evidence of previous infection is consistent with transmission by aerosol and other routes (31).
Our results showing that infectious RSV is contained in aerosolized particles are supported by two other studies that showed evidence of airborne RSV RNA as far as 7 m from infected patients. However, these studies did not determine if the RSV remained infectious (9, 14). It is important to note that in the results reported here, it cannot be concluded that all of the aerosolized particles containing RSV came from the infected infants, because during the seasonal outbreak of RSV, parents, visitors, and staff may have also been infected. However, no infectious virus was found at the nurse’s station 10 m from the bronchiolitis bay, suggesting contribution of RSV particles by staff was unlikely to be significant.
In summary, we have shown that high levels of infectious RSV is found in both large and very small aerosolized particles in the room air around infants with RSV bronchiolitis. In the UK and many other countries, due to the lack of available isolation cubicles on wards and intensive care, infants with clinically suspected bronchiolitis are frequently nursed in an open bay, often before virus identification. These data suggest these practices may need to be reviewed to reduce the nosocomial spread of RSV. The focus of our work is now to determine ways of reducing the amount of RSV contained in aerosol particles in a hospital setting.
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Supported by a Cystic Fibrosis Trust clinical fellowship and Action Medical Research project grants (SP4499 and SP4118).
Author Contributions: Contributed to study design, acquisition and analysis of data, and assembly of the manuscript: H.K. and C.M.S. Contributed to acquisition and analysis of data, and review of the manuscript: D.D.H.L. and R.A.H. Contributed to the study design, analysis of data assembly and final approval of the manuscript: A.J.E. Conceived the study, led the study design, analysis of the data, and assembly and final approval of the manuscript: C.O’C.
This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.201509-1833OC on February 18, 2016