Challenge of Liquid Stressed Protective Materials and Environmental Persistence of Ebola Virus

Abstract

After the largest Ebola virus outbreak in history, experts have attempted to answer how the Zaire ebolavirus species emerged in West Africa and caused chains of human-to-human transmission. The widespread and untimely infection of Health Care Workers (HCW) in the affected countries accelerated spread of the virus within the community. Among the reasons attributed to this trend, it must be considered that HCW were exposed to the virus in their occupational environment. The contribution of environmental conditions to the spread of Ebola in West Africa was examined by investigating the effect of temperature/humidity on the virus's environmental persistence and by modeling if saturation (liquid stress) allows for penetration of Ebola virus through personal protective equipment (PPE). Ebola-Makona virus persisted on PPE and materials found in outbreak settings for less than 72 hours at 27 °C and 80% relative humidity (RH). A difference in virus penetration was observed between dry (5%, 1/21 tests) and saturated (33%, 7/21 tests) samples of PPE. Infectious virus particles penetrated through saturated coupons of Tyvek Micro Clean, Tychem QC, whole surgical masks and N95 respirators. These findings suggest inclusion of saturation or similar liquid stress simulation in protective equipment testing standards.

Figure 1

Persistence of infectious Ebola virus and viral RNA on PPE and ETU materials. Ebola virus was recovered from materials incubated in average West African climate conditions (27 °C and 80% RH) using a variation of the QCT-2 protocol published as ASTM E2197 – 11. The Environmental Persistence experiments were performed in three biological replicates, each consisting of three technical repetitions. Reported values are the average across all replicates (n = 9) error was calculated as standard error of the mean. Where: GE- genome equivalents and TCID₅₀- tissue culture infectious dose fifty.

Figure 2

Ebola virus penetration through three types of protective material, which allowed the virus to persist for ≥24 hrs at 27 °C and 80% relative humidity. Virus was inoculated onto the surface of a dry or saturated coupon and left to incubate at 21.5 °C and 30–35% RH for 30 minutes. The coupons were then swabbed on the exposed and unexposed side to recover the virus used in the challenge and detect viral penetration through the material. Where: P- positive (inoculation/challenge) control, SR- swab recovery, D- dry sample, S-saturated sample, M- mock. The PPE Penetration experiments were performed in four biological replicates, each consisting of one technical repetition. Reported values are the average across all replicates (n = 4) error was calculated as standard error of the mean.

Figure 3

Ebola or VSV-GFP virus penetration through a surgical mask and N95 respirators challenged in dry and saturated conditions. Whole masks were fitted onto the face of a respiratory mannequin and challenged with Ebola virus. The mannequin was programmed to breath for thirty minutes at 21.5 °C and 30–35% RH. The masks were then removed and the inside surface of the mask and face of the mannequin were dry swabbed to detect virus penetration: (A) surgical mask, (B) KC 200 N95 respirator, (C) KC 300 N95 respirator. An equal volume of virus used to challenge the masks was back titrated as a positive control. Recovery of vRNA but not infectious virus particles suggest that saturation of the examined masks compromised protection by 100%. Where: DM-dry mask, DF- dry face, WM- wet mask, WF- wet face, P-positive/inoculation control, M-mock. The Mask Penetration experiments were performed in three biological replicates, each consisting of three technical repetitions. Reported values are the average across all replicates (n = 9) error was calculated as standard error of the mean. A paired T-test was used to calculate significant differences in mean values between the test and negative control (P-value: not significant >0.05, *≤0.05, **≤0.01 and ***≤0.001).