MATERIALS AND METHODS

Environmental samples were collected from the treatment rooms and the male and female patients' toilets of GUM clinics A and B on six occasions and one occasion, respectively. The various sites sampled are listed in table 1. Environmental samples were also collected from male and female toilets at a hospital based staff leisure and fitness centre on one occasion. Samples were usually obtained during the working day, although the sampling at clinic A on the fifth and sixth occasion was performed at 8.30 am and 4.30 pm of the same day.

Samples were collected using cotton wool tipped swabs soaked in 0.1M phosphate buffered saline (PBS) pH 7.2. All environmental surfaces were swabbed firmly in order to pick up any cells that may have been present and gloves were changed between each sample. Samples were assigned a number on the arrival at the laboratory in order to mask the source of each individual sample during testing. Only after the testing of all samples was performed were laboratory numbers correlated with the sample source.

Serial dilutions of CaSki cells, containing 600 copies HPV-16 DNA per cell (starting concentration 4 × 10⁶ cells/ml) and RNase-free water were included as positive and negative controls, respectively. Nucleic acids were extracted from all samples and controls using the guanidinium isothiocyanate/silica procedure.¹⁰

All samples were tested for the presence of β globin DNA using a real time PCR (LightCycler; Roche Diagnostics, UK), with the GH20/PC04 primers¹¹ before they were tested for HPV DNA, using a single tube nested real time PCR,¹² in order to determine the sample quality. The presence of β globin DNA was determined by melting the PCR product in order to measure its melting temperatures (Tm). The Tm values of the reaction products were compared to that of a positive control. A minimum of 15 copies of β globin DNA could be detected using this method.

HPV DNA was detected using the LightCycler, with the MY09/MY11¹³ and GP5+/GP6+ primers¹⁴ in a single tube nested PCR.¹² The presence of HPV DNA was determined by melting the PCR product in order to measure its Tm. A minimum of 10 HPV DNA input copies could be detected through the use of the single tube nested PCR.

Two microlitres, of HPV DNA positive, nested PCR amplicons were used to repeat the second round of the PCR with the GP5+ fluorophore labelled primer¹⁵ and the GP6+ primer in order to generate products for HPV typing. These PCRs were carried out on a conventional block based thermal cycler (Perkin Elmer, GenAmp 2400) as described previously.¹⁶

Reverse line hybridisation was performed as described by Jordens et al.¹⁵ Probes complementary to sequences of the L1 region of HPV types 2, 6, 11, 16, 18, 31, 33, 35, 39, 41, 42, 43, 45, 50, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 67, 70, 72, Han 831, and CP8304 were used.

RESULTS

HPV DNA was detected in environmental samples collected from GUM clinic A on more than one occasion. HPV DNA was found mostly associated with surfaces of treatment areas and equipment used for the assessment and treatment of patients, although HPV DNA was also found in both the male and female patients' toilets (table 1). β Globin DNA was detected in all HPV DNA positive samples, and was not detected in any of the HPV negative samples collected from GUM clinic A. HPV DNA was not detected in any of the samples collected from GUM clinic B or from the staff leisure and fitness centre (table 2). β Globin DNA was not detected in any of the samples from the staff leisure and fitness centre and was found in only three samples from GUM clinic B (table 2).

Table 3 shows the accumulation of HPV types throughout GUM clinic A during a single day. At 8.30 am HPV DNA was detected only in samples from the female and male toilets, and on the surface of the cryotherapy guns. In samples collected at 4.30 pm, HPV DNA was detected throughout the clinic, toilets, and on the equipment used for the assessment and treatment of patients (Table 3).

A total of 19 HPV types, all associated with genital HPV infection, were detected on environmental surfaces. HPV types 6, 16, and 11 were the most commonly found types, accounting for 28.6%, 24.2%, and 9.8%, respectively, of all HPV types detected in the environment. These correspond to the distribution and prevalence of HPV types identified from genital swabs of GUM clinic patients on different occasions (unpublished data).

DISCUSSION

The aim of this study was to investigate the possibility of environmental contamination with genital HPV types in “high risk” and “low risk” environments.

HPV infected cells were shown to be present on many of the surfaces within the treatment rooms in GUM clinic A. The spectrum of HPV types detected was similar to that found in the attendees of this GUM clinic (unpublished data).

The failure to type four of the HPV amplicons may have been due to the absence of a specific probe, complementary to sequences in the PCR amplicon. The probes used were specific for fewer than 50% of known HPV types, although they were chosen to match the spectrum of mainly genital HPV types detected in the population of the United Kingdom.

All identified HPV types detected in the treatment rooms were mucosal HPV types, ruling out the possibility of contamination from cutaneous wart virus. HPV DNA detected on the bed control panel, light switch, and the colposcope handle were much more likely to indicate contamination from the doctor's gloved hands after he/she had examined the patient. Contamination of the examination bed and of the patients' toilets is more likely to have arisen from the patients themselves, as these are areas with which medical and nursing staff have little or no contact. The toilets sampled were used exclusively by patients, and all HPV types detected were of mucosal origin indicating transfer from genital sites to the environment via hands or by direct contact. HPV DNA was detected at a higher frequency in the male toilets then in the female toilets of GUM clinic A, and only two HPV types were detected in the female toilets compared to five HPV types in the male toilets, again suggesting the transfer of HPV DNA was from the genitalia via the hands onto the environmental surfaces. Hand carriage of genital HPV DNA has been shown in a previous study, where patients with genital HPV infections were found to carry the same HPV type on their hands.⁹

The cleaning regimes employed in the clinics and the space available for treating patients were factors associated with the presence or absence of contamination. GUM clinic A had no formal cleaning procedures in place, whereas GUM clinic B had established very rigorous and strict cleaning procedures. The patients' toilets of clinic A were cleaned once a day by the contract cleaning staff. Treatment rooms, as well as equipment, were only cleaned at the end of each week, with the exception of the examination bed, which was cleaned at the end of each day. GUM clinic A houses two small treatment rooms and one male and female toilet adjacent to the waiting area. GUM clinic B was very spacious, with six new treatment rooms, each with their own adjacent patient toilet.

In GUM clinic A equipment used, such as cryotherapy guns, was frequently moved between treatment rooms and was never cleaned. In GUM clinic B, individual cryotherapy spray guns were used less frequently, owing to greater use of self treatment, and were allocated to each room and after use were wiped down with Azowipe (70% isopropyl alcohol BP, Vernon-Carus Ltd, UK). Interestingly, eight out of the 14 HPV types detected on the cryoguns of clinic A were HPV types which were never detected on any environmental surfaces within the clinic. All these eight HPV types (43, 45, 51, 58, 59, 67, 70, Han 831) are associated with cervical abnormalities or neoplasia rather then clinically obvious lesions.

The number of clinic attendees in GUM clinic A was higher than that of clinic B, resulting in the throughput for each individual room being higher for GUM clinic A than GUM clinic B. Out of a total of approximately 100 patients attending GUM clinic A per day up to 20 had genital warts. However, in GUM clinic B a total of 19 patients, of which only three patients had warts and four patients attended for colposcopy, were seen on the day of sampling. On the day before sampling a total of 25 patients, of whom three had genital warts, were seen in GUM clinic B.

Samples obtained before and after working hours in GUM clinic A showed an absence of HPV and β globin DNA before patients attending. This suggests degradation of cellular and HPV DNA which had contaminated surfaces on the previous working day.

The detection of HPV DNA does not necessarily indicate the presence of viable virus. The lack of in vitro culture procedures for HPV at present does not allow the viability of the virus to be determined. The correlation between the detection of HPV DNA and β globin DNA would suggest that only cell associated viral DNA was recovered. Bovine papillomavirus type 1 has been shown to survive desiccation in cell extracts for up to 7 days.³

The quantity of virus present on environmental surfaces was not determined. However, the single tube real time nested PCR enables the detection of a minimum of 10 input copies of HPV DNA; therefore, even a very small number of HPV DNA present could have been recovered from the sites sampled in clinic A. It is possible that even if viable virus was present this may be insufficient to infect an epithelial site by hand transfer. Given that the possibility of transmission via environmental surfaces is neither confirmed nor disproved, precautions should be taken to prevent the transfer of infected cells via human “vectors.” Regular cleaning of the clinic environment and equipment would reduce the potential for infected cells to contaminate environmental surfaces. It is likely that the more rigorous cleaning regime of GUM clinic B, together with fewer patient attendances was the reason for the absence of detectable β globin and HPV DNA. GUM clinic A has implemented a more rigorous cleaning policy since we undertook this study, and we hope to re-examine the issue of environmental viral contamination in the near future.

CONTRIBUTORS

The principal author, SS, with the co-author, PS, collected the samples, and performed the PCR and the reverse hybridisation on the environmental samples; CS supervised the sample collection in GUM clinic A and was co-author; SE supervised the sample collection in GUM clinic B and was co-author; JG supervised the project and was senior author.

Funding was provided by the Public Health Laboratory Service for whom the Cambridge laboratory acts as the National Human Papillomavirus Reference Laboratory.