Infectious titres of human papillomaviruses (HPVs) in patient lesions, methodological considerations in evaluating HPV infectivity and implications for the efficacy of high-level disinfectants

Abstract

Background: Recent publications from a single research group have suggested that aldehyde-based high-level disinfectants (HLDs), such as ortho-phthalaldehyde (OPA), are not effective at inactivating HPVs and that therefore, patients may be at risk of HPV infection from medical devices. These results could have significant public health consequences and therefore necessitated evaluation of their reproducibility and clinical relevance.

Methods: We developed methods and used standardised controls to: (1) quantify the infectious levels of clinically-sourced HPVs from patient lesions and compare them to laboratory-derived HPVs, (2) evaluate experimental factors that should be controlled to ensure consistent and reproducible infectivity measurements of different HPV genotypes, and (3) determine the efficacy of select HLDs.

Findings: A novel focus forming unit (FFU) infectivity assay demonstrated that exfoliates from patient anogenital lesions and respiratory papillomas yielded infectious HPV burdens up to 2.7 × 10³ FFU; therefore, using 2.2 × 10² to 1.0 × 10⁴ FFU of laboratory-derived HPVs in disinfection assays provides a relevant range for clinical exposures. RNase and neutralising antibody sensitivities were used to ensure valid infectivity measures of tissue-derived and recombinant HPV preparations. HPV infectivity was demonstrated over a dynamic range of 4-5 log₁₀; and disinfection with OPA and hypochlorite was achieved over 3 to >4 log₁₀ with multiple genotypes of tissue-derived and recombinant HPV isolates.

Interpretation: This work, along with a companion publication from an independent lab in this issue, address a major public health question by showing that HPVs are susceptible to HLDs.

Funding: Advanced Sterilization Products; US NIH (R01CA207368, U19AI084081, P30CA118100).

Keywords: High-level disinfectant; Medical devices; Nosocomial infection; Oncogenic virus; Sexually transmitted disease; Virus disinfection.

Fig. 1

Clinical lesion sampling and quantification of HPV genome load from samples. (a) Exfoliating cells and squames were collected from the surface of clinically-diagnosed HPV lesions using five passes of a sterile emery paper (see Supplemental Information). Image made in collaboration with BioRender. (b) DNA was isolated from exfoliated cells from atop clinically-diagnosed papillomas and subject to genotyping. Samples positive for HPV11, HPV16 and/or HPV31 were assessed for genotype specific viral DNA load in triplicate by qPCR. Also shown are the estimated sum levels of genome copies per sample for non-HPV11 low risk types (types 6, 43, 44, 54, 61, 70) or non-HPV16/31 high-risk types, (types 33, 35, 39, 53, 56, 59, 68). Bars represent the mean values of total VGE from each sample; mean values and 95% CI are shown. Details of cases are shown in Table 1.

Fig. 2

Infection time course and antibody neutralisation characteristics of HPV virion stocks. Replicate cultures of subconfluent HaCaT cells were incubated with recombinant HPV quasivirions (a, c, e) or tissue-derived HPV preparations (b, d, f). Inocula were exposed to cells for 5 min and were either harvested for total RNA (0h infection), or fresh media were added for infection for 24h or 48 h. A replicate set for each virus stock was incubated with a genotype-specific anti-virus-like particle, monoclonal antibody prior to cell exposure for 48h. The antibodies included: anti-HPV11 clone 11F.G1, anti-HPV16 clone H16.V5, anti-HPV31 clone H31.A6. (g, h) Raft tissue-derived virus stocks were untreated or RNase A treated prior to cell exposure for 0 or 48 h. RNAs were subjected to RT and triplicate qPCR for spliced E1^E4 mRNA quantification (circles); values were normalised to 100% infection at 48 h p.i. Bars represent the mean values of triplicate qPCR reactions from two experimental replicate infections; error bars indicate SD [with paired, two-tailed t-tests performed on indicated values].

Fig. 3

Linear dose response dynamic range of RT-qPCR quantification and infectivity titration of infectious HPV stocks. HaCaT cells were exposed to serial dilutions of HPV virus stocks that were validated as in Fig. 2. At 48 h p.i., total RNA was extracted and subjected to RT-qPCR with triplicate copy number controls from 10⁸ to 10¹ for quantification of HPV E1^E4; ß-actin was a reference target. The y-axis represents the total number of E1^E4 cDNA copies present in triplicate amplifications relative to internal calibration curves attaining ≥95% efficiency. The limit of detection (LoD) for each calibration curve is indicated for each qPCR series. Bars represent the mean of qPCR triplicates (symbols) for each infection replicate; SD error bars are shown. Hashtags (#) specify samples with no detectable E1^E4 targets. Simple linear regression of the mean triplicate values (R²) is shown for each replicate dilution series.

Fig. 4

Infectivity of HPV-positive clinical samples compared with laboratory-sourced HPVs. Genotyped clinical samples from recurrent respiratory papillomas (RRP) and anogenital warts (AW) were freeze-thawed 3x prior to exposure to subconfluent HaCaT cells for 48 h. Xenograft and QV HPV stocks and mock-infected samples were included as positive and negative controls, respectively. (a-b). RT-qPCR with copy number controls from 10⁷ to 10¹ for quantification of HPV E1^E4 as in Fig. 2. (c-n) Cells were seeded on chamber slides and exposed to HPV stocks. RNA in situ hybridisation (RNA-ISH) was performed for high-risk (HR) and low-risk (LR) HPV genotypes (see Supplemental Information). (c-e) Controls showing specificity of RNA-ISH as labeled. RNA-ISH was performed for LR HPV11 in lab and clinical HPV stocks (f-j) and HR RNA-ISH for HPV16 in lab and clinical HPV stocks (k-n) with focus forming unit (FFU) comparisons to the inocula dose (VGE) for each assay. Arrows point to positive cells containing ≥2 positive puncta indicative of HPV E6/E7 mRNA. Bars = 100 µm unless otherwise noted.

Fig. 5

Disinfection efficiency of OPA and hypochlorite against validated HPV stocks. According to the schematic in Suppl. Fig. 2, 5 µL of virus stocks (infection validated as in Fig. 2) were incubated with buffer as a control or disinfectants (OPA or hypochlorite [ClO-]) for 12min. Virus-disinfectant and virus control (buffer) solutions were neutralised and subject to 10-fold serial dilutions in neutraliser and incubated 15 min (final virus dilutions included 10-²–10⁻⁶). Virus was also incubated with neutralised disinfectants (light pink bars) or in cell medium (control; dark pink bars). (a-c) 1% glycine was the OPA neutraliser; (d-f) 7% glycine was the OPA neutraliser. As indicated, virus was incubated with dilutions of a neutralising monoclonal antibody (NAb; striped pink bars). HaCaT cells were exposed to each virus stock for 48 h; the physical load (VGE) and infectious load (FFU) of each virus in the 10⁻² dilution is indicated. Infectivity levels were determined by RT-qPCR for HPV E1^E4 mRNAs compared to intra-assay, E1^E4 cDNA internal calibration curves; bars represent the mean values of triplicate qPCR reactions. Hashtags (#) specify samples with no detectable E1^E4 targets and the limit of detection (LoD) for each qPCR assays is indicated. Log reduction (yellow boxes) was determined by comparing disinfectant-treated virus to virus treated with buffer (black lines). Neutralised disinfectant and antibody effects on virus infection were compared to untreated control virus (red lines).