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The spread of influenza is of increasing concern due to its pandemic potential. Influenza virus can be transmitted via contact, large droplets, and aerosol routes. The relative contribution of different routes is still unknown and is under debate. Some researchers believe that large droplet transmission is the dominant route, while others[2, 3] argue in favor of aerosols. That the aerosol route is an important mode of transmission is clear from recent studies.[4-9]
One common way to reduce the spread of influenza is to use personal protective equipment (PPE) such as face masks, respirators, and garments, which are often made of non-woven fabrics. However, there is concern that influenza virus may survive on PPE long enough to render used PPE into vehicles for virus transmission. The virus could be easily transferred from contaminated PPE to the skin when the PPE is removed from healthcare workers, which may greatly increase the risk of contact transmission. It has also been documented that influenza virus can survive on a wide variety of surfaces including non-woven fabrics.[11-15]
The above-mentioned studies utilized spiking with virus as a challenge method; that is to apply a known concentration of virus suspension onto surfaces of interest followed by virus elution. The comparative efficacy of different eluents used in these studies has not been investigated. One reason for poor virus recovery from porous surfaces is believed to be inefficient elution of the virus. Therefore, in studies on virus survival on PPE and their subsequent transfer from PPE, an optimum eluent for virus recovery should be found by comparing the recovery efficiency of different eluents.
Another limitation of the previous studies is that the test methods relying on virus spike tests may not sufficiently mimic the real-life situation of aerosol or large droplet transmission of influenza, although they may serve as good proxies to simulate contamination of fomites. As reviewed by Gralton et al., breathing, coughing, sneezing, and talking can easily generate particles of a wide size range, and influenza virus has been detected in the particles generated by human respiratory activities.[8, 17, 18] Therefore, we investigated the difference in viable influenza virus recovery from PPE after liquid suspension spike tests versus aerosol challenge tests.
The objectives of this study were (i) to evaluate the efficiency of eight eluents for recovering influenza virus from three non-woven fabrics commonly used to manufacture PPE and (ii) to compare influenza virus recovery from non-woven fabrics spiked with virus suspension versus loaded with virus aerosol.
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The recovery efficiency of frequently used eluents[12-15] was systematically evaluated using spike tests. Although no statistically significant differences were observed in the recovery efficiency of the eight eluents, the type of non-woven material (e.g., hydrophobic or hydrophilic) was found to be a major factor influencing recovery of AIV, which should be noted in future studies involving the recovery of spiked virus from surfaces.
Virus recovery efficiency of spike tests can be best viewed by looking at recovery data at the drying time of 0 min with recovery less than 100% in most cases. Similar results were previously reported, showing only 10% of the applied influenza virus recovered from a serge before drying took place. With minimum virus inactivation by desiccation at zero drying time, any decrease of virus titer could be attributed to virus adsorption onto the non-woven fabrics. One recent study found that the recovery efficiency of H5N1 influenza virus spiked onto a polypropylene respirator was 70 ± 5% as determined by quantitative PCR, further suggesting that virus adsorption onto surfaces could affect virus recovery.
Virus adsorption onto surfaces can be reduced using electrostatic repulsion, which depends on the isoelectric point (IEP) of the virus and the surface as well as pH of the eluent. The IEP for the three non-woven fabrics tested is listed in Table 1 and that of influenza virus ranges from 4·6 to 5·4. Therefore, at eluent pH of 7·0 to 9·0, both the non-woven fabric and the influenza virus carried net negative surface charges and repelled each other, aiding virus recovery from the surfaces. Theoretically, there may be an increase in repulsive force with increased difference between IEP of virus or non-woven fabric and the eluent pH. However, the monotonic increase of recovery efficiency with increased pH was only observed for PP and PET using 3% BE and for Nylon using 1·5% BE at a drying time of 0 min. The high IEP of Nylon may partially explain the low recovery efficiency of spiked virus compared with PP and PET.
Hydrophobic interaction was expected to be dominant in the attachment of lipid-containing virus (e.g., influenza virus) onto hydrophobic surfaces (e.g., PP and PET). However, the hydrophobic fabrics gave a much higher virus recovery than hydrophilic Nylon, which agrees with the work of Sakaguchi et al. The AIV suspension formed droplets when applied to hydrophobic PP and PET surfaces with a small contact area between the virus and fibers while it easily spread and soaked into the Nylon fabrics with a much larger contact area and a higher probability for virus adsorption onto the fibers, consequently lowering the recovery efficiency. Dissolved organic matter such as protein tends to reduce hydrophobic interaction. However, the increase of BE from 1·5% to 3% did not increase virus recovery, especially for Nylon (Figure 2). Increased BE concentration was also found to yield a lower recovery of MS2 bacteriophage and the mechanisms behind it are not clear.
A significant decrease in recovery with increased drying time might be due to a combined effect of inefficient virus removal from non-woven fabric and inactivation of virus by desiccation. The wind draughts in the biosafety cabinet might also result in virus loss from the fabric surface to the cabinet air, especially after long drying time. It is possible that virus was brought closer to the non-woven surface and became more easily adsorbed onto the fabric as the virus suspension evaporated (as in the case of Nylon at a drying time of 0 min), thus reducing virus recovery. On the other hand, inactivation of influenza virus on surfaces by desiccation is well known. The stress-sensitive nature of influenza virus makes it difficult to determine whether the physical removal of viable virus from non-wovens is a function of drying time.
An optimum eluent to recover virus from PPE should dislodge virus effectively from the surface and help maintain virus viability once it gets removed. Optimum eluents could be designed for each specific virus-surface pair by adjusting the eluent pH and concentration of organic composition. Adding surfactants such as NaPP and Tween 80 may help by minimizing hydrophobic interactions. In addition, inclusion of chaotropic and monovalent salts in the eluent can also promote virus recovery by decreasing the ordering of water molecules. Certain PPE such as N95 respirators generally contains electrostatically charged (electret) fabrics, which may enhance virus adsorption onto surfaces. Ethanol has been used to recover virus from electret filter media, because it can degrade the charge of fabrics and help elute more virus. However, it should be noted that electret-containing PPE may become less effective after ethanol treatment.
Conventional protocols that use spike tests as a virus challenge method may at best simulate fomite and contact transmission of influenza. In this study, AIV was applied to non-woven fabrics in the form of aerosols, which better simulated the airborne transmission of influenza. Although neither the type of eluent nor the non-woven fabric was found to significantly affect virus recovery, the results indicated that AIV recovery significantly depended on how the virus was applied (Table 2). Recovery of aerosolized virus was much more difficult than that of spiked virus, suggesting spike tests cannot be simply taken as an approximation for aerosol challenge tests in studies of virus survival on surfaces where airborne transmission of influenza virus may get involved.
The low recovery of aerosolized influenza virus from non-woven fabrics is comparable to a 3·2% recovery from respirators found in another study. Compared to spike tests, the lower recovery found in aerosol tests could be due to the poorer survivability of influenza virus in aerosol particles than in liquid suspension.[2, 32] Another reason could be the inefficient physical removal of virus from non-wovens. The generated submicron virus aerosol particles could easily diffuse and be deposited deep into the non-woven fabric layer, making them difficult to recover. For example, electron microscopy analysis has shown that many particles remained on filter fibers after extraction by vortexing. This may explain why there was no significant difference in recovery between hydrophobic and hydrophilic surfaces as seen in the spike tests.
One of the limitations of this study is that the agitation method used in the spike tests (orbital shaking) was different from that used in the aerosol challenge tests (vortexing). The different amplitude and frequency of the agitation method may cause different relative acceleration motion between eluent and non-woven fabrics, yielding different recovery results. Nevertheless, Fisher et al found that sonication, vortexing, and shaking exhibited similar efficiency and repeatability for extracting aerosolized virus from respirator coupons, suggesting agitation methods gave minimal difference in virus recovery efficiency. Second, volume of the eluent and area of the non-woven fabric used in aerosol challenge tests were larger than spike tests (e.g., 49 cm2 versus 1 cm2 and 5 ml versus 1 ml), leading to different volume to area ratios in the two tests (~0·1 ml/cm2 versus 1·0 ml/cm2). A low volume to area ratio might result in insufficient mixing/elution and yield lower virus recovery in the aerosol challenge tests. Both the area of the fabric and the volume to area ratio should be standardized across the two tests to enable better comparison. Third, temperature and humidity during the spike tests should have been controlled, because they may affect the determination of drying time.
One should be cautious to generalize the experimental results to real-life situations. First, the nebulizer suspension tested was different from the suspending environment for the naturally aerosolized virus. In reality, virus may be encased within particles containing mucin and other respiratory excretion substances, the presence of which has been demonstrated to extend the survival of influenza virus and probably gives higher virus recovery. Second, PPE in real life could be contaminated by both contact and aerosolized particles, and therefore, the actual virus recovery may fall between what determined from the spike and the aerosol tests. Third, only one layer of fabric was tested while PPE could be made of multiple layers of electret fabrics, which may significantly affect virus recovery. Fourth, the drying time and challenge time tested might be short compared with clinical settings, where PPE may be worn for up to three hours. Therefore, the virus recovery at the time of PPE doffing (when virus transmission most likely to happen) may be different from what determined in this study. Last, although low virus recovery was found in the aerosol tests, in real life all used PPE should be regarded as contaminated and be removed appropriately.
For future studies, it will be helpful to use quantitative RT-PCR to measure total (both viable and non-viable) virus recovered from non-wovens, given the fact that infectious influenza virus is rarely found in natural environments due to its extremely low concentration. Although RT-PCR provides no indication of virus infectivity, it has been found to give a higher rate of virus detection than culture methods.[17, 18, 34, 35] RT-PCR can be used combined with culture methods to help differentiate the relative contribution to recovery efficiency by the inefficient physical removal of virus from the non-wovens and by the natural decay of virus infectivity (e.g., desiccation). With the virus recovery efficiency issue sorted out, it will be interesting to investigate the potential difference in influenza virus survival kinetics on PPE when it is applied as a liquid suspension versus an aerosol.