Dynamics of papillomavirus in vivo disease formation & susceptibility to high-level disinfection-Implications for transmission in clinical settings

Abstract

Background: High-level disinfection protects tens-of-millions of patients from the transmission of viruses on reusable medical devices. The efficacy of high-level disinfectants for preventing human papillomavirus (HPV) transmission has been called into question by recent publications, which if true, would have significant public health implications.

Methods: Evaluation of the clinical relevance of these published findings required the development of novel methods to quantify and compare: (i) Infectious titres of lab-produced, clinically-sourced, and animal-derived papillomaviruses, (ii) The papillomavirus dose responses in the newly developed in vitro and in vivo models, and the kinetics of in vivo disease formation, and (iii) The efficacy of high-level disinfectants in inactivating papillomaviruses in these systems.

Findings: Clinical virus titres obtained from cervical lesions were comparable to those obtained from tissue (raft-culture) and in vivo models. A mouse tail infection model showed a clear dose-response for disease formation, that papillomaviruses remain stable and infective on fomite surfaces for at least 8 weeks without squames and up to a year with squames, and that there is a 10-fold drop in virus titre with transfer from a fomite surface to a new infection site. Disinfectants such as ortho-phthalaldehyde and hydrogen peroxide, but not ethanol, were highly effective at inactivating multiple HPV types in vitro and in vivo.

Interpretation: Together with comparable results presented in a companion manuscript from an independent laboratory, this work demonstrates that high-level disinfectants inactivate HPV and highlights the need for standardized and well-controlled methods to assess HPV transmission and disinfection.

Funding: Advanced Sterilization Products, UK-MRC (MR/S024409/1 and MC-PC-13050) and Addenbrookes Charitable Trust.

Keywords: Cervical cancer; HPV, nosocomial transmission; High-level disinfectant; Methodology for virus infection assay; Virus disinfection.

Fig. 1

Variation in human papillomavirus VGE titres at the surface of the cervix (a) Exfoliating cells from 40 patients were collected from the surface of the cervix using a nitrocellulose patch (see Methods). (b) After Benzonase digestion to remove non-encapsidated viral DNA, quantitative HPV typing was carried to determine virus type and titre (VGE/mm²; Y-axis). For each patient, associated disease was classified either as LSIL (blue), HSIL (red), or as encompassing both HSIL and LSIL together (yellow). p = 0.058, LSIL versus HSIL (Mann Whitney test). (c) Detail of HPV types and VGE abundance in LSIL and HSIL are shown. Virus titre in the exfoliating cervical cells of the cervix of 40 different patients are shown as VGE/mm2. HPV types detected at the lesion surface are listed, with the most prominent HPV type in each case highlighted in red and virus titres.

Fig. 2

Quantification of virus infectious titre in vitro (a, b) Measurement of E1^E4 viral gene transcripts or Gaussia luciferase reporter gene activity in HaCaT cells infected with differing doses of HPV18 (red), MmuPV1 (green) and PsV (black). Infectious titre shown with 5-6 log₁₀ dynamic range. Neutralising assay compares virus titre following incubation with the neutralising antibody or isotypic control. AU, arbitrary unit; NT, not tested; ND, not detected. Data is obtained with biological triplicates and shown as Mean and SD. (c, d) RNAscope® visualisation of E6 and E7 viral gene transcripts in HaCaT cells infected with HPV18 (top) or MmuPV1 (bottom); virus titre shown as VGE/cell; proportion of virus-RNA positive cells (cell boundary in red) given as %.; boxed regions enlarged in (d). Scale bar; 100 μm

Fig. 3

Quantification of virus infectivity in vivo using MmuPV1 model (a) Proportion of lesions formed at infected sites over a 16-week post-inoculation observation period with differing virus doses. * indicates p < 0.05, **indicates p < 0.01 (the log-rank test). The numbers of sites of inoculation (N) and mice in each group are shown. (b) Number of weeks until appearance of first lesion post-inoculation at differing virus doses are shown as Mean and Range. The numbers of sites of inoculation and lesion formed are shown. (c) Proportion of lesions formed at infected sites over a 16-week post-inoculation observation period for either virus with exfoliating cells on fomite (kept for 0, 8 and 52 weeks) or cell-free virus on fomite (kept for 0 and 8 weeks). Fig. 3a results are shown for comparison, in grey. **indicates p < 0.01 (the log-rank test). The numbers of sites of inoculation (N) and mice in each group are shown.

Fig. 4

Viral titration assay results provide a scale to interpret each read-out of virus titration in the context of VGE, infectious titre and actual capability of in vivo lesion formation.

Fig. 5

Evaluation of disinfectant efficacy using in vitro infection assay (a, b) Measurement of viral infectivity (E1^E4 viral gene transcripts or reporter gene activity shown as Mean and SD) of HPV18, MmuPV1 and PsV in HaCaT cells following incubation with viruses treated with disinfectants or their neutralised equivalent (except 70% ethanol). AU, arbitrary unit; ND, not detected. Data were obtained with biological triplicates and shown as Mean and SD.

Fig. 6

OPA-glycine disrupts the electrostatic charge of virus particles and inhibits infection (a) Virus infectivity (E1^E4 viral gene transcripts or reporter gene activity) of HPV18, MmuPV1 and PsV in HaCaT cells following incubation with OPA-glycine and OPA-lysine. ND, not detected. (b) Schematic representation of the reaction of OPA with glycine and lysine. Estimated pI and charge at pH 7.4 are shown. (c) Relative amount of cation-exchange (cation) or anion-exchange (anion) membrane-sequestered OPA-glycine and OPA-lysine during ion-exchange chromatography. AU, arbitrary unit. (d) Measurement of infectious dose? of membrane-sequestered PsV, and PsV plus OPA-glycine or OPA-lysine. (e) Relative VGE of membrane-sequestered HPV18 and MmuPV1 in the presence of OPA-glycine or OPA-lysine. All data were shown as Mean and SD and obtained with biological triplicates.

Fig. 7

Evaluation of efficacy of disinfectants using MmuPV1 in vivo infection assay Proportion of lesions formed at infected sites over a 16-week observation period following inoculation with cell-free MmuPV1 previously incubated with (a) disinfectant or (b) neutralised disinfectant, or MmuPV11 with exfoliating cells on fomite previously incubated with (c) disinfectant or (d) neutralised disinfectant. The numbers of sites of inoculation (N) and mice in each group are shown.