The ability of two chlorine dioxide chemistries to inactivate human papillomavirus‐contaminated endocavitary ultrasound probes and nasendoscopes

Abstract 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.


| INTRODUCTION
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, 1 attributable to large numbers of cancers of the cervix, uterus, anus, and head and neck. 2,3 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 Prevention. 4,5 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  stably maintaining HPV episomes were cultured in E-medium with J2-3T3 feeder cells and grown in raft culture to produce a virus, as previously described. 22,23 Mature virus particles were harvested from tissues after 20 days. [24][25][26] Rafts were harvested and the virus was isolated by homogenization in phosphate buffer (5 mM Naphosphate; pH 8; 2 mM MgCl 2 ), as previously described. 22,23 All virus 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 cellular debris.

| Virus titers
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 Kit. 23 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 8 to 10 4 copies per mL. 23 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.

| Instrument preparation
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.

| Disinfectants
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 hypochlorite 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 carrier tests. 14,15 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.

| Disinfection procedure
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.

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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 described. 15 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-qPCRbased infectivity assay for E1^E4 transcript levels. 23 The E1^E4 spliced transcript was amplified using primers specific for the spliced transcript. HPV16 and 18 infectivity assays were performed using HaCat cells, as previously described. 22,23 Complete viral inactivation was considered achieved when post disinfection infectivity assays showed equivalent or higher C t values than uninfected controls.

| RESULTS
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 10 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). 14

| DISCUSSION
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 demonstrates that oxidizing chemistries, including hydrogen peroxide, peracetic acid blended with silver, sodium hypochlorite, 13,15,27 and now chlorine dioxide, are effective at inactivating both HPV16 and HPV18.
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. F I G U R E 1 Susceptibility of HPV16 and HPV18 virions to chlorine dioxide disinfectants. A total of 1 × 10 7 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 10 reduction of infectivity for each condition tested. HaCat cells were used for the infectivity assays. The dotted line marks the FDA required 4 log 10 reductions. FDA, Food and Drug Administration Furthermore and more importantly, it provides a solution to