Foi feita uma avaliação da eficácia do vapor de peróxido de hidrogênio a 35% (HPV) contra o vírus da febre aftosa (FMDV) em uma instalação de biossegurança. A experimentação mostra que o vírus da febre aftosa é suscetível a descontaminação por HPV, se tornando uma alternativa potencial ao formaldeído.

1. ORIGINAL ARTICLE
Evaluating the efficacy of hydrogen peroxide vapour
against foot-and-mouth disease virus within a BSL4
biosafety facility
B.M. Petit2
, F.C. Almeida1
, T.R. Uchiyama1
, F.O.C. Lopes1
, K.H. Tino2
and J. Chewins3
1 INOVA Biotecnologia, Juatuba, Minas Gerais, Brazil
2 STEQ Com e Representacß~oes LTDA, Sao Paulo, Brazil
3 Bioquell UK Limited, Andover, UK
Significance and Impact of the Study: Foot-and-mouth disease virus (FMDV) is an important pathogen
in terms of biosafety due to its infectious nature and wide range of host animals, such as cattle, sheep,
goats and pigs. Outbreaks of FMDV can have a severe impact on livestock production, causing morbid-
ity, mortality, reduced yields and trade embargoes. Laboratories studying FMDV must possess BSL4
robust bio-decontamination methods to prevent inadvertent release. Formaldehyde has been the pri-
mary agent for environmental decontamination, but its designation as a human carcinogen has led to a
search for alternatives. This study shows 35% hydrogen peroxide vapour has the potential to be a rapid,
effective, residue-free alternative.
Keywords
biosecurity, decontamination, foot-and-mouth
disease, hydrogen peroxide, virus.
Correspondence
John Chewins, Bioquell UK Ltd, Andover,
Hampshire SP10 4AR, UK.
E-mail: John.Chewins@Bioquell.com
2017/0932: received 13 May 2017, revised 6
July 2017 and accepted 6 July 2017
doi:10.1111/lam.12778
Abstract
An evaluation was made of the efficacy of 35% hydrogen peroxide vapour
(HPV) against foot-and-mouth disease virus (FMDV) in a biosafety facility.
Biological indicators (BIs) were produced using three serotypes of FMDV, all
with a titre of ≥106
TCID50 per ml. Fifteen BIs of each serotype were
distributed across five locations, throughout a 30-m3
airlock chamber,
producing a total of 45 BIs. Thirty-five percent HPV was generated and
applied using a Bioquell vaporization module located in the centre of the
chamber. After a dwell period of 40 min, the HPV was removed via the
enclosures air handling system and the BIs were collected. The surfaces of the
BIs were recovered into Glasgow’s modified Eagle’s medium (GMEM),
cultivated in BHK21 Cl13 cell culture and analysed for evidence of cytopathic
effect (CPE). No CPE was detected in any BI sample. Positive controls showed
CPE. The experimentation shows that FMDV is susceptible to HPV
decontamination and presents a potential alternative to formaldehyde.
Introduction
Foot-and-mouth disease virus (FMDV) is often referred
to as the animal plague (FMDV, 2016), classified as
requiring mandatory notification and included in the OIE
(World Organisation for Animal Health) Group 4 list of
pathogens. It is a highly infectious virus of the family
Picornaviridae and affects cloven-hoofed mammals,
including key livestock animals such as goats, pigs, sheep
and cattle, and a group of nondomestic animals. In 2001,
an FMDV outbreak occurred within the United Kingdom,
resulting in mass culls of livestock. The UK National
Audit Office estimated the costs of the outbreak to be £3
billion to the public sector and £5 billion to the private
sector (UK NAO, 2016). Laboratories working with
FMDV must have robust biosafety measures in place to
prevent the inadvertent release of the virus to the envi-
ronment, where it could colonize wildlife and subse-
quently spread into livestock. A key part of any biosafety
protocol is the decontamination of equipment and materi-
als from contaminated areas where virus manipulation has
occurred to clean areas via transfer devices such as airlocks.
Formaldehyde has been used as the primary decontamina-
tion agent for environmental decontamination within
Letters in Applied Microbiology 65, 281--284 © 2017 The Society for Applied Microbiology 281
Letters in Applied Microbiology ISSN 0266-8254

2. high-level biosafety facilities, recognized and approved by
authorities. In June 2014, formaldehyde was reclassified in
Europe as a Class 1B carcinogen and Class 2 mutagen. The
US National Toxicology Program describes formaldehyde
as ‘known to be a human carcinogen’. This classification
along with other disadvantages associated with formalde-
hyde fumigation (such as long exposure times and resi-
dues) is driving the search for an efficacious,
noncarcinogenic alternative. Hydrogen peroxide vapour
(HPV) decontamination has been used within pharmaceu-
tical and health care facilities for many years. There is a
wealth of scientific literature supporting the efficacy of
35% HPV systems (Hall et al. 2007; Boyce et al. 2008;
Otter et al. 2010; Goyal et al. 2014; Lemmen et al. 2015;
Holmandahl et al. 2016), including comparative head-to-
head studies with formaldehyde (Beswick et al. 2011) and
exotic viruses (Heckert et al. 1997). However, specific effi-
cacy data against FMDV is lacking. The aim of this evalua-
tion is to determine whether FMVD can be eliminated
using 35% HPV.
Results and discussion
Forty-five biological indicators produced using FMDV
cultures of ≥106
TCID50 mlÀ1
(Tissue Culture Infective
Dose) produced no cytopathic effect (CPE) when intro-
duced to monolayers of BHK21 Cl13 cells after exposure
to 35% HPV for a period of 115 min in three replicate
cycles (15 BIs per cycle to give a total of 45 BIs) con-
ducted over three separate days. BIs were located in the
corners and centre of a 30-m3
airlock at both ceiling
and floor level validating efficacy throughout the entire
chamber (see Fig. 1). The three FMDV serotypes used –
O1 Campos, A24 Cruzeiro and C3 Indaial – showed full
inactivation in all three replicate tests, indicated by the
lack of CPE and complement fixation. These serotypes
were used because, in Brazil, companies that produce
FMDV vaccine are only authorized to handle these sero-
types. Positive controls for the three serotypes were used
and showed CPE when introduced to BHK21 Cl13 cell
cultures. Negative controls exposed to the HPV cycle
showed no adverse toxicological effect when introduced
to cell cultures.
The results correspond with previous work conducted
with swine vesicular disease virus (SVDV) another mem-
ber of the Picornaviridae family (Heckert et al. 1997).
SVDV was eliminated when exposed to HPV for 30 min.
This suggests that the 115 min exposure used in this
experimentation may be in excess of that required to
achieve kill and further studies should be conducted to
understand better the time point at which inactivation
occurs.
The results of this preliminary experimentation suggest
that 35% HPV is able to achieve full inactivation of
FMDV on a hard surface within a room on a repeatable
basis. Use of formaldehyde within the same enclosure
requires a contact time of 600 min (10 h), thus the use of
35% HPV, even with a potentially excessive cycle of
115 min, confers substantial advantages in terms of effi-
ciency. HPV breaks down to form oxygen and water,
leaving no residues within the chamber. In conclusion,
this early stage work identifies 35% HPV to be a possible
alternative to formaldehyde for the bio-decontamination
of facilities that handle FMDV and confers advantages
over and above formaldehyde in terms of speed, human
toxicity and residues. Further work is required to evaluate
alternative surfaces and the impact of interfering sub-
stances such as organic material.
A limitation of the study is that the positive control
was stored in the fridge and thus the reduction in the
virus attributable to exposure to room temperature was
not quantified.
Materials and methods
A Bioquell Clarus R vaporization module (STEQ Servicßos,
S~ao Paulo, Brazil) was located in the centre of a 30-m3
sealed airlock (airlock number 222) within a BSL4 Unit
approved for FMDV handling (INOVA Biotecnologia,
Juatuba – Minas Gerais, Brazil) by the Brazilian Ministry
of Agriculture, Livestock and Supply. Bioquell HPV-AQ
35% hydrogen peroxide (STEQ Servicßos) was placed into
the vaporization module. The temperature and humidity
were controlled using the facilities HVAC, with the tem-
perature ranging between 19°C and 25°C during the test-
ing (humidity was not recorded).
External door
HPV
generator
Internal
door
Airlock
54
3
2
1
Figure 1 Locations of foot-and-mouth disease virus biological indica-
tors (FMDV BIs) (three strains per location) within the airlock. BIs were
placed on the ceiling (4), the floor (2), opposing corners of the room
(1, 5) and on the HPV generator (3).
Letters in Applied Microbiology 65, 281--284 © 2017 The Society for Applied Microbiology282
Evaluating HPV against FMDV B.M. Petit et al.

3. Virus suspensions were produced through the infection
of BHK21 Cl13 cells in Glasgow’s modified Eagle’s med-
ium (GMEM), without bovine serum. Viral titration was
carried out in 96-well plates, using BHK21 Cl13 cells in
compliance with the Brazilian Ministry of Agriculture,
Livestock and Supply Regulatory Procedure (BRASIL,
Ministerio da Agricultura, 2008). Viral suspensions pro-
duced positive complement fixation results for typing and
thus, based on the complement fixation and titration
results, were determined to be infectious with ≥106
TCID50 mlÀ1
. Six FMDV BIs were produced for each ser-
otype O1 Campos, A24 Cruzeiro and C3 Indaial. To pro-
duce a BI, 100 ll of viral suspension (107 8
TCID50 mlÀ1
)
was inoculated onto the inside surface of a polypropylene
cryogenic storage tube cap (TPP Techno Plastic Products,
Trasadingen, Switzerland) and left to completely dry in a
Class 2 biological safety cabinet (BSC). Five BIs for each
FMDV serotype were placed into the airlock containing
the HPV generator at the locations indicated in Fig. 1.
One BI per serotype was retained as a positive control
and stored in a refrigerator at 2–8°C for the duration of
the exposure experiment. A negative control was pro-
duced by applying 100 ll of GMEM to the inside surface
of a cryotube cap and allowing it to completely dry. The
negative control was placed inside the airlock at location
3 (see Fig. 1).
The hermetic doors were sealed and the HPV cycle initi-
ated. The HPV generator injected HPV for 75 min, fol-
lowed by a 40-min dwell period, after that aeration to
1Á0 ppm. Samples were contained and carefully removed
from the airlock. In accordance with current regulatory
requirements and standard operating procedures, the airlock
was exposed to formaldehyde fumigation prior to re-use.
BHK21 Cl13 cell culture monolayers were grown in
roller bottles for 48 h. Three roller bottles were produced
per test BI, plus one bottle for each positive control and
one bottle for the negative control. Evaluation of residual
active virus was carried out in accordance with Brazilian
Ministry of Agriculture, Livestock and Supply Regulatory
Procedure (BRASIL 2008). The samples of HPV-exposed
virus were recovered from the caps via a quantitative
washing process using GMEM and aseptically transferred
to the prepared cell monolayer within the roller bottles.
Three-hundred millilitre of sterile GMEM cultivation
media was added to each roller bottle. This process was
repeated for all BI samples and the positive and negative
controls. Roller bottles were incubated in a roller shelve
oven at 35–37°C for 24 h.
Bottles were analysed macroscopically and microscopi-
cally for CPE and compared to the negative control. If
CPE was identified, the supernatant was removed and
subject to complement fixation analysis. If no CPE was
detected, the process was continued for a second and
third passage and incubated for 48 h. At the end of the
passage process, cell cultures showing no CPE were sub-
mitted to complement fixation analysis to confirm the
negative result.
The experiment was repeated to produce three replicate
cycles.
Conflict of Interest
B. Petit and K. Tino are employed by STEQ Servicßos a
provider of HPV decontamination equipment and ser-
vices. J. Chewins is an employee of Bioquell UK Limited,
a manufacturer of HPV generators. F.C. Almeida, F.O.C.
Lopes and T.R. Uchiyama have no conflicts to declare.
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