Influenza Aerosols Research Articles

Influenza virus survival in aerosols and estimates of viable virus loss resulting from aerosolization and air-sampling

Using a Collison nebulizer, aerosols of influenza (A/Udorn/307/72 H3N2) were generated within a controlled experimental chamber, from known starting virus concentrations. Air samples collected after variable suspension times were tested quantitatively using both plaque and polymerase chain reaction assays, to compare the proportion of viable virus against the amount of detectable viral RNA. These experiments showed that whereas influenza RNA copies were well preserved, the number of viable viruses decreased by a factor of 10(4)-10(5). This suggests that air-sampling studies for assessing infection control risks that detect only influenza RNA may greatly overestimate the amount of viable virus available to cause infection.

An experimental investigation of the performance of a Collison nebulizer generating H1N1 influenza aerosols

The performance of a Collison nebulizer delivering viable H1N1 influenza aerosols was assessed in terms of particle size distribution (PSD) and survivability of the virus upon generation. An H1N1 influenza virus preparation in egg allantoic fluid was diluted in sterile deionized water to a concentration of 3.4 × 106 TCID50/mL. The virus suspension was aerosolized at air flow rates of 2, 6 and 12 L/min using a 1-jet, 3-jet and 6-jet Collison nebulizer, respectively. A scanning mobility particle sizer measured the PSD of the viral aerosol after steady-state delivery times of 1, 15, 30, 45 and 60 min. After 60 min of continuous aerosolization, the viral titre was unchanged and the count median diameter (CMD) of the aerosol PSD was ∼38 nm for the 2 L/min flow rate, ∼35 nm for the 6 L/min flow rate and ∼33 nm for the 12 L/min flow rate. The CMDs were much smaller than the influenza virus (80–120 nm), indicating the aerosol distribution comprised mainly nonviable materials. The PSD produced by the Collison nebulizer exhibited a 20% increase in peak particle concentration after 60 min of continuous operation at 12 L/min. This progressive increase in particle counts may be attributed to a combination of evaporation and shear and impact stresses imparted on components by the Collison nebulizer. The possible slight loss in H1N1 influenza viability over the course of 60 min of continuous aerosolization at 12 L/min is consistent with previous bioaerosol studies using a Collison nebulizer.

Influenza aerosols in UK hospitals during the H1N1 (2009) pandemic--the risk of aerosol generation during medical procedures.

BackgroundNosocomial infection of health-care workers (HCWs) during outbreaks of respiratory infections (e.g. Influenza A H1N1 (2009)) is a significant concern for public health policy makers. World Health Organization (WHO)-defined ‘aerosol generating procedures’ (AGPs) are thought to increase the risk of aerosol transmission to HCWs, but there are presently insufficient data to quantify risk accurately or establish a hierarchy of risk-prone procedures.Methodology/Principal FindingsThis study measured the amount of H1N1 (2009) RNA in aerosols in the vicinity of H1N1 positive patients undergoing AGPs to help quantify the potential risk of transmission to HCWs. There were 99 sampling occasions (windows) producing a total of 198 May stages for analysis in the size ranges 0.86–7.3 µm. Considering stages 2 (4–7.3 µm) and 3 (0.86–4 µm) as comprising one sample, viral RNA was detected in 14 (14.1%) air samples from 10 (25.6%) patients. Twenty three air samples were collected while potential AGPs were being performed of which 6 (26.1%) contained viral RNA; in contrast, 76 May samples were collected when no WHO 2009 defined AGP was being performed of which 8 (10.5%) contained viral RNA (unadjusted OR = 2.84 (95% CI 1.11–7.24) adjusted OR = 4.31 (0.83–22.5)).Conclusions/SignificanceWith our small sample size we found that AGPs do not significantly increase the probability of sampling an H1N1 (2009) positive aerosol (OR (95% CI) = 4.31 (0.83–22.5). Although the probability of detecting positive H1N1 (2009) positive aerosols when performing various AGPs on intensive care patients above the baseline rate (i.e. in the absence of AGPs) did not reach significance, there was a trend towards hierarchy of AGPs, placing bronchoscopy and respiratory and airway suctioning above baseline (background) values. Further, larger studies are required but these preliminary findings may be of benefit to infection control teams.

Influenza aerosols in UK hospitals during the influenza H1N1 (2009–10) pandemic: air sampling in the vicinity of patients undergoing WHO-defined aerosol generating procedures

BackgroundNosocomial infection of health-care workers during outbreaks of respiratory infections (eg, influenza A H1N1 [2009]) is a great concern for public health policy makers. WHO-defined aerosol generating procedures (AGPs; eg, bronchoscopy, non-invasive ventilation) are thought to increase risk of aerosol transmission to health-care workers, but data to quantify risk accurately are insufficient. This study aims to provide some evidence base behind the requirement for respirators with FFP2 protection level to be worn in the vicinity of patients infected with influenza. MethodsWe measured the amount of pandemic H1N1 (2009) RNA in aerosols in the vicinity of H1N1-positive patients undergoing AGPs to help to quantify the potential risk of transmission to health-care workers. Sampling was done in the 2009–10 influenza seasons in five hospitals in England. Peak sampling times corresponded with peak levels of influenza activity (Nov, 2009–Jan, 2010; and Nov, 2010–Jan, 2011). Patient inclusion criteria were new changes in chest radiograph in the presence of one or more of: central temperature 38°C or higher (38·5°C in children) or 36°C or lower, white blood cell count less than 4000 or more than 12–000 cells per μL, positive microbiology or virology from respiratory secretions, and mucopurulent secretions from the respiratory tract. Aerosol sampling was done with the May 3 stage impinger (which allows fractionation of air samples into three aerodynamic size ranges: 0·86–4·0, 4·0–7·3, and >7·3 μm) at head height about 1 m away from the patient. Air samples were taken while WHO-defined AGPs were occurring, and baseline samples were taken when no AGP was being done. Samples were returned frozen to the laboratory, concentrated by centrifugation, and assayed by quantitative rt-PCR. A sample was deemed positive for aerosolised influenza particles if influenza RNA was detected in particles in the aerodynamic size range 0·86–7·3 μm. We used a univariable logistic regression model, adjusted for repeated measurements on the same individual, to examine the relation between propensity to obtain a positive H1N1 aerosol sample and undertaking of AGPs. Exact binomial confidence intervals were calculated for unadjusted sample proportions. FindingsWe obtained 198 air samples from 39 patients with influenza H1N1 (2009) during 99 sampling windows. Most participants were male (25 of 39 [64%]) and older than 50 years (28 of 39 [72%]). Only four (10%) participants were younger than 5 years. Mean time of sampling was 5·5 days (SD 8·56) since diagnosis. Viral RNA was detected in 21 of 198 (10·6%, 95% CI 6·7–15·8) air samples from ten of 39 (26%) patients. 46 air samples were obtained while WHO-defined AGPs were being done, of which nine (19·6%, 95% CI 9·4–33·9) contained viral RNA. Of 152 samples taken when no AGP was being done, 12 (7·9%, 4·2–13·4) contained viral RNA. Although AGPs did not significantly increase the probability of sampling an H1N1 (2009) positive aerosol, the point estimate and associated 95% CI from the regression model indicated that risk is likely to be heightened (odds ratio 4·31, 95% CI 0·83–22·5). InterpretationOur findings show that H1N1 RNA aerosols were often associated with infected patients within intensive-care units and were more frequently detected when a WHO-defined AGP was being done. Our results should be interpreted with caution because of the small sample size and limitations of the study design, which could not control for all potential sources of bias. Nevertheless on the basis of our results, we believe that infection control guidance for the use of FFP2 respirators in the vicinity of H1N1-positive patients should stand, until further data can be obtained. FundingHealth Protection Agency.

Detection of Airborne Influenza A Virus in Experimentally Infected Pigs With Maternally Derived Antibodies

This study assessed whether recently weaned piglets with maternally derived antibodies were able to generate infectious influenza aerosols. Three groups of piglets were assembled based on the vaccination status of the dam. Sows were either non-vaccinated (CTRL) or vaccinated with the same (VAC-HOM) strain or a different (VAC-HET) strain to the one used for challenge. Piglets acquired the maternally derived antibodies by directly suckling colostrum from their respective dams. At weaning, pigs were challenged with influenza virus by direct contact with an infected pig (seeder pig) and clinical signs evaluated. Air samples, collected using a liquid cyclonic air collector, and individual nasal swabs were collected daily for 10 days from each group and tested by matrix real-time reverse transcriptase polymerase chain reaction (RRT-PCR) assay. Virus isolation and titration were attempted for air samples on Madin-Darby canine kidney cells. All individual pigs from both VAC-HET and CTRL groups tested positive during the study but only one pig in the VAC-HOM group was positive by nasal swab RRT-PCR. Influenza virus could not be detected or isolated from air samples from the VAC-HOM group. Influenza A virus was isolated from 3.2% and 6.4% air samples from both the VAC-HET and CTRL groups, respectively. Positive RRT-PCR air samples were only detected in VAC-HET and CTRL groups on day 7 post-exposure. Overall, this study provides evidence that recently weaned pigs with maternally derived immunity without obvious clinical signs of influenza infection can generate influenza infectious aerosols which is relevant to the transmission and the ecology of influenza virus in pigs.

Development of a murine nose-only inhalation model of influenza: comparison of disease caused by instilled and inhaled A/PR/8/34

Influenza continues to cause widespread disease and death during winter months. In preclinical studies to evaluate the potential efficacy of drugs and vaccines, influenza challenge virus is usually instilled into the noses of animals in the form of large liquid drops. Since inhalation of aerosolized influenza is commonly associated with human transmission, instillation of challenge virus raises uncertainty about the applicability of results. In order to compare the challenge methods, we established conditions to generate influenza aerosols with a mass median aerodynamic diameter (MMAD) of 1 μm that were delivered to mice in a nose-only inhalation system. In this report, we describe the system and compare the 50% lethal dose (LD50) of instilled and inhaled A/PR/8/34 (PR8) in BALB/c mice. The estimated LD50 for inhaled virus was 8.7 plaque forming units (PFU) and the mean time to death was 7.7 days, whereas the estimated LD50 for instilled virus was 51.6 PFU and the mean time to death was 8.2 days. Our results show that mice are more sensitive to inhaled virus than virus delivered by intranasal instillation. The murine nose-only inhalation model of influenza infection can be used to infect large numbers of animals simultaneously with well-characterized, homogenous PR8 bioaerosol in a controlled and reproducible manner. This model provides the means to evaluate the efficacy of drug and vaccine candidates against the relevant route of challenge, thereby providing data that may better predict clinical outcome.

Protective effect of low-concentration chlorine dioxide gas against influenza A virus infection

Influenza virus infection is one of the major causes of human morbidity and mortality. Between humans, this virus spreads mostly via aerosols excreted from the respiratory system. Current means of prevention of influenza virus infection are not entirely satisfactory because of their limited efficacy. Safe and effective preventive measures against pandemic influenza are greatly needed. We demonstrate that infection of mice induced by aerosols of influenza A virus was prevented by chlorine dioxide (ClO(2)) gas at an extremely low concentration (below the long-term permissible exposure level to humans, namely 0.1 p.p.m.). Mice in semi-closed cages were exposed to aerosols of influenza A virus (1 LD(50)) and ClO(2) gas (0.03 p.p.m.) simultaneously for 15 min. Three days after exposure, pulmonary virus titre (TCID(50)) was 10(2.6+/-1.5) in five mice treated with ClO(2), whilst it was 10(6.7+/-0.2) in five mice that had not been treated (P=0.003). Cumulative mortality after 16 days was 0/10 mice treated with ClO(2) and 7/10 mice that had not been treated (P=0.002). In in vitro experiments, ClO(2) denatured viral envelope proteins (haemagglutinin and neuraminidase) that are indispensable for infectivity of the virus, and abolished infectivity. Taken together, we conclude that ClO(2) gas is effective at preventing aerosol-induced influenza virus infection in mice by denaturing viral envelope proteins at a concentration well below the permissible exposure level to humans. ClO(2) gas could therefore be useful as a preventive means against influenza in places of human activity without necessitating evacuation.

Role of interferon in mice in protection against influenza A virus by bacterial ribosomes together with membranal glycoproteins of Klebsiella pneumoniae as adjuvant.

Nonspecific protection against infectious aerosols of influenza A virus was obtained in Swiss mice after vaccination by aerosols of bacterial ribosomes together with membranal glycoproteins extracted from Klebsiella pneumoniae as the adjuvant. It was shown that repeated stimulant aerosols were necessary to obtain this protection. Routine estimation of serum interferon levels after administration of the association of ribosomes plus membranal glycoproteins to the animals by aerosol or intravenous route showed that there was no correlation between protection and the presence of serum interferon. It was shown that the serum interferon-inducer activity was due to ribosomes. No induction of serum interferon was obtained with membranal glycoproteins used separately. Local liberation of interferon in the mucous membrane of the upper respiratory tract was not investigated.

Inhibition of Respiratory Virus Infections of Mice with Aerosols of Synthetic Double-Stranded Ribonucleic Acid

Aerosols of double-stranded complexes of polyinosinic and polycytidylic acids (poly I:C) were useful in protecting mice infected with aerosols of influenza (A(2)/Taiwan/64) and parainfluenza type 1 (Sendai) viruses. Administration of poly I:C as an aerosol offers an advantage, particularly in therapy, by eliminating the risk of pulmonary dissemination of viral infections due to intranasally instilled fluids. Treatment of mice with aerosols of poly I:C reduced the infection rate with influenza virus but did not inhibit virus multiplication in the lungs of most of those animals where infection became established. Sendai virus infection rates were undiminished in mice treated with poly I:C, but lung-virus titers were significantly suppressed as compared with those of untreated animals. The maximum poly I:C doses (40 mug) administered by aerosol produced no evidence of toxicity in the mice.

Studies on air-borne virus infections: III. The killing of aerial suspensions of influenza virus by hypochlorous acid

Experiments are described for determining the effect of hypochlorite mists and hypochlorous acid gas on aerosols of influenza A virus.The virus aerosol is shown to be at least as easily killed by hypochlorite mists as Streptococcus salivarius. It is also killed by liberating hypochlorous acid gas itself into the atmosphere. A concentration of 1 vol. of gas in 2 million vol. of air is probably effective in destroying 99% or more of virus particles when the proportion of these in the air is small.Preliminary experiments on mice and cats are recorded which failed to reveal any toxic effects produced by inhaling the gas in relatively high concentrations or for prolonged periods. Acute irritation of mucous membranes only was found. This did not appear to lead to any increased susceptibility of mice to subsequent infection with influenza.We wish to acknowledge our indebtedness to the late Sir Patrick Laidlaw and to Dr R. J. V. Pulvertaft in co-operation with whom the earlier experiments were made. We also wish to thank Dr W. J. Elford, Dr F. C. MacIntosh, Dr R. K. Callow and Dr T. S. Work for their assistance and advice in parts of the investigation.