© 2020 The Authors. Journal of Medical Virology published by Wiley Periodicals, Inc.
J Med Virol. 2020;1–5. wileyonlinelibrary.com/journal/jmv
Received: 6 November 2019
Accepted: 7 January 2020
The ability of two chlorine dioxide chemistries to inactivate
human papillomavirus‐contaminated endocavitary
ultrasound probes and nasendoscopes
Department of Microbiology and
Immunology, Pennsylvania State College of
Medicine, Hershey, Pennsylvania
Department of Microbiology and Molecular
Biology, Brigham Young University, Provo,
Craig Meyers, Department of Microbiology
and Immunology, Pennsylvania State College
of Medicine, Hershey, PA 17036.
Tristel Solutions Limited, UK
Sexual transmission is the most common pathway for the spread of Human
papillomavirus (HPV). However, the potential for iatrogenic HPV infections is also
real. Even though cleared by the Food and Drug Administration and recommended by
the World Federation for Ultrasound in Medicine and Biology, several disinfectants
including glutaraldehyde and o‐phthalaldehyde have shown a lack of efficacy for
inactivating HPV. Other methods such as ultraviolet C and concentrated hydrogen
peroxide have been shown highly effective at inactivating infectious HPV. In this
study, two chlorine dioxide systems are also shown to be highly efficacious at
inactivating HPV. An important difference in these present studies is that as opposed
to testing in suspension or using a carrier, we dried the infectious virus directly onto
endocavitary ultrasound probes and nasendoscopes, therefore, validating a more
realistic system to demonstrate disinfectant efficacy.
antiviral agents, dissemination, human papillomavirus, immune responses, papillomavirus,
pathogenesis, reinfection, virus classification
Human papillomavirus (HPV) is a small, nonenveloped DNA virus
with over 200 types identified. These types are classified as either
high‐risk for their implication in cancers in areas such as the cervix,
uterus, and head and neck, or low‐risk types, which cause benign
condylomas or warts. Types 16 and 18 are classified as high‐risk and
are documented to be the most prevalent types worldwide,
attributable to large numbers of cancers of the cervix, uterus, anus,
and head and neck.
Sexual transmission via oral or penetrative
means is widely documented in the scientific literature and is
highlighted for its risk by healthcare institutions such as the National
Health Service and the Centers for Disease Control and Preven-
However, a source of potential transmission via fomites in the
healthcare environment from inadequate disinfection practices has
become an area of concern, debate, and discussion. Clinical areas in
which examination, diagnoses, or treatment is provided through the
use of instruments entering body cavities, cavities where HPV16 and
18 are prevalent, pose a risk to clinician and patient. Obstetrics,
gynecology, and emergency medicine departments are examples of
areas where devices including transvaginal endocavity ultrasound,
colposcopes, and speculums are used to examine the cervix and can
subsequently be contaminated with HPV.
such as endoscopes used within otorhinolaryngology departments
are also at risk of HPV contamination.
The World Federation for Ultrasound in Medicine and Biology
decontamination guidelines for transvaginal ultrasound transducers
recommends disinfectants that include: 2.4% to 3.2% glutaraldehyde
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
(GTA), o‐phthalaldehyde (OPA), 7.5% hydrogen peroxide, 0.5%
bleach, ultraviolet C (UVC) radiation at 200 to 280 nm and chlorine
Our previous work in which testing was performed with
UVC radiation at 253.7 nm, 0.525% and 0.87% bleach, and 31.5%
sonicated hydrogen peroxide, has demonstrated the efficacy of these
treatments in inactivating HPV16 and 18.
Where only HPV16
was tested, disinfection was not achieved with 0.55% OPA, or 2.4%
or 3.4% GTA.
We, therefore, considered the next logical step for
testing would entail the assessment of chlorine dioxide, as
recommended in the guidelines noted above. These chlorine dioxide
products have been referenced in otorhinolaryngology disinfection
guidelines such as ENT UK
and of the Official Journal of the Italian
Society of Otorhinolaryngology.
Published studies show the use of
chlorine dioxide products across the globe in countries including the
UK, Australia, New Zealand, and Singapore.
describes the testing of two chlorine dioxide products to determine
their ability to adequately disinfect devices contaminated with HPV.
Here, we used a different approach for testing the two chlorine
dioxide solutions against HPV16 and HPV18 vs. our previous studies,
which assessed efficacy in suspension or carrier‐based assays. In this
study, we contaminated actual medical devices, endocavitary ultra-
sound probes, and nasendoscopes, with the virus to simulate in‐use
disinfection as closely as possible.
MATERIALS AND METHODS
Cell culture and virus production
HaCaT cells were maintained in Dulbecco's modified Eagle's medium
DMEM supplemented with 10% fetal bovine serum (FBS), 0.025 mg/
mL gentamicin, and 0.11 mg/mL sodium pyruvate. Primary human
keratinocytes from newborn foreskin circumcision were isolated, as
The Human Subjects Protection Office of
the Institutional Review Board at Penn State University College of
Medicine screened our study design for exempt status according to
institutional policies and the provisions of applicable federal
regulations. They determined this study did not require formal IRB
review because no human participants are involved as defined by
federal regulations. Keratinocytes were maintained in 154 medium
supplemented with a Human Keratinocyte Growth Supplement Kit
(Cascade Biologics Inc, Portland, OR). Immortalized keratinocytes
stably maintaining HPV episomes were cultured in E‐medium with
J2‐3T3 feeder cells and grown in raft culture to produce a virus, as
Mature virus particles were harvested
from tissues after 20 days.
Rafts were harvested and the virus
was isolated by homogenization in phosphate buffer (5 mM Na‐
phosphate; pH 8; 2 mM MgCl
), as previously described.
preparations for concentration and infectivity assays were treated
with Benzonase (375 U) at 37°C for 1 hour to remove any
unencapsidated viral genomes. Samples were adjusted to 1M NaCl
and centrifuged at 4°C for 10 minutes at 10 500 rcf to remove
To release the viral genomes, 10 mL of a virus preparation was
resuspended in 200 mL HIRT DNA extraction buffer (400 mM NaCl/
10 mM Tris‐HCl, pH 7.4/10 mM EDTA, pH 8.0), with 2 mL 20 mg/mL
Proteinase K, and 10 mL 10% sodium dodecyl sulfate for 2 hours at
37°C. The DNA was purified by phenol‐chloroform extraction followed
by ethanol precipitation and resuspension in 20 mL TE. Titers were
determined using a quantitative polymerase chain reaction (qPCR)‐based
DNA encapsidation assay utilizing a Qiagen Quantitect SYBR Green PCR
Amplification of the viral genome target was performed using the
previously described E2 primers against a standard curve of 10‐fold
serial dilutions from 10
copies per mL.
For infection assays,
HaCaT cells were seeded in 24‐well plates with 50 000 cells per well
2 days before infection. Compounds were mixed with virus and media in
a total volume of 500 µL before addition to cells. An multiplicity of
infection (MOI) of 10 particles per cell was used unless otherwise noted.
The virus was incubated with the cells for 48 hours at 37°C and
messenger RNA was harvested using a Qiagen RNAeasy Kit.
Instruments tested were (a) nasendoscopes and (b) endocavity
ultrasound probes. An organic load (soil) of 5% FBS was added to
the virus suspension and spread along the length of the insertion
tube of each device, representing the part of the instrument exposed
to the patient. The inoculated instruments were allowed to dry in a
laminar flow cabinet for 30 minutes or until dry.
The two chlorine dioxide disinfection procedures used were from Tristel
Solutions Limited: (a) the Tristel Trio Wipes System and (b) Tristel Duo.
The ability of each procedure to inactivate authentic HPV16 and 18 was
evaluated separately. As a positive disinfection control, sodium hypo-
chlorite was used at the manufacturer's recommended concentration of
0.87% (8700 parts per million) (Pure Bright Germicidal Ultra Bleach, KIK
International). The use of this control was based on its previously
demonstrated efficacy against HPV16 and 18, in both suspension and
To control for virus recovery after drying onto the
probe, some probes were not treated with disinfectant and the virus was
removed and tested for infectivity, as described below. All disinfectant
products were used according to the manufacturer's instructions for use.
The endocavity ultrasound probe and nasendoscope were disinfected
using a three‐step Tristel Trio Wipes System. This included a preclean
wipe to clean the instruments, a sporicidal wipe to disinfect the
instrument with a contact time of 30 seconds, and a rinse wipe to
MEYERS ET AL.
remove any chemical residue. This procedure replicates the standard
decontamination guidelines for semicritical medical devices, which
includes a cleaning step, a disinfection step, and a rinsing step.
The second set of endocavity ultrasound probes (Siemens)
was disinfected by first using a preclean wipe to replicate the
removal of ultrasound gel from a sheath that would be present on a
device after a clinical procedure. The device was then disinfected
with two aliquots of Tristel Duo applied via a low linting Duo Wipe,
utilizing a 30 second contact time for efficacy.
Nasendoscopes (Karl Storz Medical Supplies) were also used for
testing and were similarly treated with Tristel Duo and the Duo Wipe,
except no initial cleaning procedure, was performed. The omission of the
cleaning step was to replicate a worst‐case scenario wherein the cleaning
step may be missed, or if soiling remained on the device post‐cleaning.
After the procedures, a base neutralizer (7% glycine) was used
to rinse and scrape 2X the chlorine dioxide treated instruments,
after which they were washed 2X with phosphate‐buffered saline
(PBS) to dilute any residues of chlorine dioxide left and halt
further action. All samples were filtered and washed with HaCat
cell media 3X and assayed for infectivity as previously de-
All disinfection efficacy tests were conducted in
triplicate with separate batches of the virus.
HPV infectivity assay
Infection was analyzed using a previously described RT‐qPCR‐
based infectivity assay for E1^E4 transcript levels.
spliced transcript was amplified using primers specific for the
spliced transcript. HPV16 and 18 infectivity assays were
performed using HaCat cells, as previously described.
Complete viral inactivation was considered achieved when post
disinfection infectivity assays showed equivalent or higher
values than uninfected controls.
The chlorine dioxide solutions were able to produce a >99.99%
reduction in infectivity of HPV16 and 18 with soil (5% BSA) included
in the assays (Figure 1). The reduction is similar to that seen with
0.87% sodium hypochlorite. The differences seen in the log
reduction values between the tests with the same virus type and
between virus types reflect different starting titers.
The efficacy of chlorine dioxide on HPV16 was similar to that of
sodium hypochlorite in our previous study, the difference is that the
previous testing was performed in a suspension‐based assay, mixing
the virus with the disinfectant in solution and not by applying the
virus directly onto the devices, as we have done in this present study.
But it did allow us to determine the differences in efficacy between
different chemical groups: alcohols (ethanol, isopropanol), aldehydes
(GTA, OPA), phenol and oxidizing agents (PAA‐silver, sodium
hypochlorite, chlorine dioxide).
In this study, we report the first results of two procedures simulating
in‐use disinfection of native HPV16‐and HPV18‐contaminated
devices. These findings support our previous work, which demon-
strates that oxidizing chemistries, including hydrogen peroxide,
peracetic acid blended with silver, sodium hypochlorite,
now chlorine dioxide, are effective at inactivating both HPV16 and
These results show that a manual procedure can be used to
disinfect HPV‐contaminated devices that may not withstand methods
that utilize submersion, heat, or radiation. The endocavity ultrasound
probes (Siemens) and nasendoscopes (Karl Storz Medical Supplies)
used for our study are representative of these devices with each
device having their unique curves, ridges, and cavities that can affect
the appropriate disinfection.
Log10 Reducon of HPV16 Infecon
Log10 Reducon of HPV18 Infecon
FIGURE 1 Susceptibility of HPV16 and HPV18 virions to chlorine
dioxide disinfectants. A total of 1 × 10
HPV16 (A) or HPV18 (B) particles
were mixed with organic soil (5% FBS) and dried onto the nasendoscope
(Nas) or transvaginal (TV) ultrasound probes. Two different chlorine
dioxide disinfection procedures were tested; Tristel Duo (Duo) and
Tristel Trio Wipes (Trio), As a control for infectious virus recovery,
HPV16 and HPV18 were mixed with soil and dried onto probes, but no
disinfection procedure was included. Hypochlorite was included as a
positive control for disinfection efficacy. Graphs show log
infectivity for each condition tested. HaCat cells were used for the
infectivity assays. The dotted line marks the FDA required 4 log
reductions. FDA, Food and Drug Administration
MEYERS ET AL.
Furthermore and more importantly, it provides a solution to
those devices that are also mobile/transportable, such as those used
within the community setting by healthcare practitioners. In these
scenarios, a transportable, simple method that achieves disinfection
efficacy in short contact time, is sorely needed.
Medical devices for examination/diagnoses that can be used in a
high patient throughput manner and be transported easily are
becoming more prevalent in the healthcare industry, especially in
developing countries. A good example of this is mobile colposcopy.
These devices are used to examine the cervix and determine any
abnormal cells or precancerous lesions that may be present. These
same countries are less likely to be able to afford an automated
disinfection system, and an easily transported, non‐machine‐based
system for disinfection would be of great benefit.
HPV is a nonenveloped virus, which has demonstrated resistance
to many disinfectants, including those which are Food and Drug
Administration cleared for high‐level disinfection (GTA, OPA).
Current guidelines require high‐level disinfection of ultrasound
probes used in semicritical applications including procedures that
may involve contact with mucous membranes or broken skin.
definition, high‐level disinfection refers to the complete elimination
of all viruses and microorganisms, with the exception of bacterial
endospores, some of which are permitted to remain.
Some devices make close contact with the patient in areas in
which HPV is prevalent, and studies have demonstrated that
colposcopes are contaminated with HPV DNA, as are the glove
boxes used by medical practitioners.
Although DNA detection does
not necessarily indicate the presence of viable and infective
microorganisms, the work of M'Zali et al
showed that HPV virions
remain present on ultrasound devices used in women's healthcare,
following standard disinfection protocols. This indicates that stan-
dard protocols are inadequate to properly disinfect these devices,
putting both patient and clinician at risk for HPV transmission.
In addition to those devices used in women's healthcare, devices
that enter the mucosal cavity of the head and neck are also at risk for
contamination with HPV. In the case of emergency (eg, ambulatory)
and point of use care, instruments such as those used to intubate
patients with breathing difficulties, are exposed to mucosal secre-
tions. To aid in the quick turnaround of device usage, manual
disinfection procedures could be pivotal. It may also save in overall
healthcare costs, as rapid disinfection methods would reduce
device reprocessing downtime and also reduce the number of
A steady increase in carcinomas of the head and neck has been
reported in many countries including New Zealand,
and the United States.
Presence of HPV DNA within
tumor samples has been demonstrated through PCR amplification of
specific gene sections, indicative of active HPV infection. Furthermore,
data demonstrate the percentage of male patients positive for HPV in
the carcinomas of the head and neck is higher than that of females. It is
postulated that the higher prevalence in men may be due to the higher
viral load of HPV within the vagina and cervix than on the penis.
Research from Hernandez et al
supports these findings, revealing
transmission of HPV is higher from the cervix to the penis than from
the penis to the vagina. Thus, it is possible that transmission of HPV
during oral sex of a man with a woman may be more likely to occur
than the oral sex of a woman with a man, providing a potential
explanation for the differing percentages seen. This adds another level
to the importance of controlling the potential of high contamination
rates on devices used in the head and neck area.
Craig Meyers http://orcid.org/0000-0001-8773-3976
Janice Milici http://orcid.org/0000-0001-9185-1457
Richard Robison http://orcid.org/0000-0002-4324-5169
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How to cite this article: Meyers C, Milici J, Robison R. The
ability of two chlorine dioxide chemistries to inactivate human
papillomavirus‐contaminated endocavitary ultrasound probes
and nasendoscopes. J Med Virol. 2020;1–5.
MEYERS ET AL.