Page 73 - JSOM Summer 2023

Uniform Material Inoculation and Washing TABLE 1 Washing with Detergent Followed by Rinsing Is Necessary
A series of square patches were cut from an ACU top with for Removal of Viral RNA
a size of 3-in by 5-in. Then, 400µL of SARS-CoV-2 in viral PCR Cycle Threshold
transport medium (VTM) was added to each uniform patch. SARS-CoV-2
They were incubated in a 50mL tube for 24 hours. After the SARS-CoV-2 Eluted From
24-hour incubation period, 9mL of sterile VTM was added and SARS-CoV-2 Eluted From Patch by VTM
allowed to incubate for 24 hours to remove unbound virus. Eluted From Patch into Following
Wastewater
Patch by VTM
Following the 24-hour incubation period, 400µL of the VTM Prior to Washing During Wash Washing and
Rinsing
were removed and frozen at –20°C; the inoculated patches
>40
were placed in sterile 50mL tubes. These patches containing Patch 1 24.83 23.40 (Undetected)
bound virus were subjected to one of several treatments. These
>40
treatments involved rinsing with water under low heat condi- Patch 2 27.37 26.20 (Undetected)
tions, rinsing with water under high heat conditions, rinsing SARS-CoV-2 can be removed from U.S. Army ACU/OCP material by
with water at ambient temperature, and exposure to detergent washing with detergent followed by rinsing with water at ambient
without rinsing. All detergents were commercially available temperature using vigorous agitation to simulate machine or hand
brands. The washing procedure included adding 1.5mL of de- washing. Lower PCR cycle threshold values indicate higher concentra-
tergent (commercially obtained from Arm & Hammer, https:// tions of virus. A cycle threshold value of 40 or greater is indicative of
www.armandhammer.com/) and 40mL of water to the patch. a concentration of viral particles below the level of detection by PCR.
Mechanical agitation was achieved using a laboratory vor- RNA = ribonucleic acid, ACU = Army Combat Uniform, OCP = op-
erational camouflage pattern, VTM = viral transport medium, PCR =
tex device. Each sample was vortexed (violently agitated) for polymerase chain reaction.
20 minutes, followed by 40mL of water used for each rinse
step. Following each treatment, uniform patches were placed The Effect of Laundering, Rinsing, and Low Heat on the
into clean 50mL tubes and incubated with 9mL of viral trans- Stability of SARS-CoV-2 on the Army Combat Uniform
port media for 24 hours to remove the virus for testing. Since the initial experiments were done at ambient tempera-
ture, there was a concern that the variable levels of heat that
Results might be found in various water sources may alter the results
by increasing or decreasing the effectiveness of the detergent.
The Effect of Laundering with Detergent Followed This is especially important since military personnel are often
by Rinsing with Tap Water at Ambient Temperature using canteen water or water that has been sitting in a water
on the Stability of SARS-CoV-2 Virus Particles on the buffalo. This experiment was performed in triplicate to pro-
Army Combat Uniform vide accurate results, and average CT values were recorded.
To simulate a situation in which military personnel are re- The procedure was performed as described above with the
quired to wash their uniforms with water and detergent but caveat that during the washing step, the tubes were heated
without the benefit of a washing machine or temperature con- to the following temperatures: 25°C, 30°C, and 35°C for 15
trol, an experiment was designed in which a suspension of a minutes. These tubes were vortexed for 20 minutes and then
SARS-CoV-2 in VTM was inoculated onto two patches cut re-heated for an additional 15 minutes. The washed uniform
from an OCP uniform top that had been impregnated with patches were vortexed again for 20 minutes, removed from the
permethrin. After this inoculation and a 24-hour incubation detergent solution, and placed in clean tubes containing 40mL
period at room temperature, additional VTM was added to re- of tap water at ambient temperature to serve as the rinse step.
move the unbound virus. The OCP patches were incubated for It was found that slightly lower CT values were obtained after
another 24 hours; an aliquot of the VTM was obtained to de- the wash step for all temperatures, but that viral RNA was
termine the amount of the unbound virus. Inoculated patches still undetectable after the rinse step (Table 2). This further
were next placed into clean tubes and washed with a combi- supports the concept of rinsing as an essential component of
nation of liquid detergent and ambient temperature tap water. removing viral RNA from the uniform.
Each tube was vortexed for 20 minutes to simulate the action
of mechanical washing. The washed uniform patches were re- TABLE 2 The Removal of SARS-CoV-2 Viral RNA from ACU
moved from the detergent solution and placed into clean tubes Uniforms Is Not Dependent on Ambient Temperature
containing tap water to serve as the rinse step. An aliquot of PCR Cycle Threshold
the waste detergent/water solution was taken for evaluation as
a wastewater sample. To simulate military personnel rinsing SARS-CoV-2 SARS-CoV-2
Eluted From
their uniform, the patches were submerged in the tap water SARS-CoV-2 Eluted From Patch by VTM
30 times and then vortexed for 10 minutes. The washed and Eluted From Patch into Following
rinsed uniform patches were then placed into a clean tube, and Patch Prior to Wastewater Washing and
additional VTM was added. Following a 24-hour incubation Washing During Wash Rinsing
period, the samples were tested by PCR for the presence of Sample 1 25.53 23.87 >40
viral RNA. Interestingly, it was found that viral RNA could (25°C) Undetected
be detected in the original sample, and unbound viral RNA Sample 2 27.70 25.64 >40
was detected in both the VTM from and the wastewater after (30°C) Undetected
24-hour incubations (average cycle threshold (CT) value 26.1 Sample 3 23.23 22.53 >40
and 24.8, respectively). No viral RNA could be detected after (35°C) Undetected
the wash and rinse steps were complete (average CT greater Viral RNA remained undetectable after washing with detergent and
rinsing with tap water despite the wash step being performed at tem-
than or equal to 40). This result indicates that washing and peratures ranging from 25–35°C.
rinsing are necessary for removing viral particles from the RNA = ribonucleic acid, ACU = Army Combat Uniform, VTM = viral
ACU uniform (Table 1). transport medium, PCR = polymerase chain reaction.

SARS-CoV-2 and Cleaning of Army Combat Uniforms | 71