Evaluation of Polyhexamethylene Biguanide (PHMB) as a Disinfectant for Adenovirus

Importance Swimming pools can be a vector for transmission of adenovirus ocular infections. Polyhexamethylene biguanide (PHMB) is a disinfectant used in swimming pools and hot tubs.

Objective To determine whether PHMB is an effective disinfectant against ocular adenovirus serotypes at a concentration used to disinfect swimming pools and hot tubs.

Design In vitro laboratory study.

Interventions The direct disinfecting activity of PHMB was determined in triplicate assays by incubating 9 human adenovirus types (1, 2, 3, 4, 5, 7a, 8, 19, and 37) with PHMB concentrations of 50 and 0 ppm (micrograms per milliliter) for 24 hours at room temperature to simulate swimming pool temperatures or 40^oC to simulate hot tub temperatures.

Main Outcome Measures Plaque assays were performed to determine adenovirus titers after incubation. Titers were log₁₀ converted and mean (SD) log₁₀ reductions relative to controls were calculated. Virucidal (>99.9%) decreases in mean adenovirus titers after PHMB treatment were determined for each adenovirus type and temperature tested.

Results At room temperature, 50 ppm of PHMB produced mean reductions in titers less than 1 log₁₀ for all adenovirus types tested. At 40°C, 50 ppm of PHMB produced mean reductions in titers less than 1 log₁₀ for 2 adenovirus types and greater than 1 but less than 3 log₁₀ for 7 of 9 adenovirus types.

Conclusions and Relevance At a concentration of 50 ppm, PHMB was not virucidal against adenovirus at temperatures consistent with swimming pools or hot tubs. Recreational water maintained and sanitized with PHMB can serve as a vector for the transmission of ocular adenovirus infections.

Adenoviral ocular infections (epidemic keratoconjunctivitis [EKC], follicular conjunctivitis, and pharyngeal conjunctival fever) are the most commonly occurring ocular viral infections in the world.¹ These infections are known to occur in community- and office-based outbreaks.¹ Outbreaks of adenoviral ocular infections can be detrimental to the community because of the significant patient morbidity and the socioeconomic consequences of time lost from school and work.¹ Among the community-based outbreaks, swimming pool water has been reported to be a vector for transmission.^2-10 Derrick² first showed in 1943 that swimming pools can act as vectors for the transmission of “swimming bath conjunctivitis.” The description of the patients' symptoms suggested that these cases were pharyngeal conjunctival fever.³ This observation came 10 years before the first adenovirus was isolated.¹¹ Some of these swimming pool–related outbreaks have been linked to inadequate chlorination of the swimming pool water.^5,6,9

Proper maintenance and sanitization of recreational water is essential to preventing disease transmission. Chlorine is the most common sanitizing agent used in swimming pools. However, proper chlorination of swimming pool water has been associated with eye irritation in some people.¹² Eye irritation, chlorine maintenance, and other factors have led to the development of alternate, non–chlorine-based agents to disinfect recreational water, such as swimming pools and hot tubs. One such agent is polyhexamethylene biguanide (PHMB), an effective public health biocide registered for numerous applications by the US Environmental Protection Agency under the Federal Insecticide, Fungicide, and Rodenticide Act and supported under the Biocidal Products Directive in Europe. It is unaffected by sunlight, water temperature, or pH fluctuations, and its stability allows water to be properly maintained for longer periods, generally 7 to 14 days before additional PHMB is required.¹³ The agent is also used in ophthalmology as a topical treatment for Acanthamoeba keratitis¹⁴ and is an active antimicrobial agent in several multipurpose contact lens solutions.¹⁵

The use of PHMB for swimming pool and hot tub disinfection prompted us to determine whether PHMB is an effective disinfectant against common ocular serotypes of adenovirus.

Clinical ocular adenovirus isolates of types 1, 2, 3, 4, 5, 7a, 8, and 19 were collected anonymously from patients who sought care because of typical adenoviral ocular disease at The Charles T. Campbell Ophthalmic Microbiology Laboratory at the UPMC Eye Center, University of Pittsburgh, Pittsburgh, Pennsylvania, and frozen at −70°C. The viruses are part of a retrospective clinical collection of adenovirus isolates. The isolates were deidentified and stored for diagnostic test validations. The serotypes of the adenovirus isolates were determined using serum neutralization. No clinical isolates of adenovirus type 37 (Ad37) were recovered, so the ATCC reference strain of Ad37 was used. The types tested represent the most common adenovirus types that cause ocular infections (Ad8, Ad19, and Ad37 cause EKC; Ad3, Ad4, and Ad7a cause follicular conjunctivitis and pharyngeal conjunctival fever)¹ and types that can replicate in rabbit ocular models (Ad1, Ad2, and Ad5).¹⁶

A549 Human Lung Carcinoma cells (CCL-185; ATCC) were used to prepare the adenovirus stocks and to determine the viral titers for adenovirus. The A549 cells were grown and maintained in tissue culture medium containing Eagle's minimal essential medium supplemented with 10% fetal bovine serum (Sigma Cell Culture Reagents).

The PHMB (Baquacil Sanitizer and Algistat Step 1; Arch Chemicals) was purchased from a local swimming pool vendor in the Pittsburgh area. The PHMB concentration in Baquacil is 20%.

This study was conducted using triplicate assays with initial inocula of approximately 1.0 × 10⁶ plaque-forming units (PFU)/mL of multiple clinical ocular isolates of Ad1, Ad2, Ad3, Ad4, Ad5, Ad7a, Ad19, and ATCC Ad37 and 1.0 × 10⁴ PFU/mL of Ad8. Fifty microliters of each test virus was added to duplicate sets of 2-mL screw-capped freezing tubes (Sarstedt) containing 450 μL of PHMB at a concentration of 55.55 or 0 ppm (micrograms per milliliter), prepared in tissue culture medium. This resulted in a final PHMB concentration of 50 ppm; this concentration was used as according to the manufacturer's instruction for recreational water disinfection. One set of tubes containing the virus-PHMB mixtures was incubated for 24 hours on a bench top at room temperature to simulate swimming pool conditions. The second set of tubes containing the virus-PHMB mixtures was incubated in a 40°C bench-top water bath for 24 hours to simulate hot tub conditions. After incubation, 500 μL of tissue culture medium containing 20% fetal bovine serum was added to each tube in preparation for the plaque assay.

Immediately after incubation and the addition of 500 μL of tissue culture medium, the samples were serially diluted in tissue culture medium containing 10% fetal bovine serum for five 10-fold dilutions and inoculated in duplicate onto A549 cell monolayers in 24-well multiplates. After a 3-hour adsorption period, the wells were filled with tissue culture medium containing 0.5% methylcellulose, except for Ad8, for which the wells were filled with standard tissue culture medium. After 7 to 10 days of incubation, the medium was removed, the cells were fixed and stained with 0.5% gentian violet solution containing formaldehyde, and the number of plaques were counted with a dissecting microscope. The viral titers were calculated and expressed as PFU per milliliter.

Viral titers were log₁₀ converted, and log₁₀ differences in titers from the appropriate negative controls were calculated for each trial. The mean (SD) log₁₀ differences in titers were calculated from the data of the 3 trials. A mean reduction in titer of at least 3 log₁₀ (99.9% reduction) relative to negative control values was considered a virucidal reduction.

The mean log₁₀ reductions from 3 trials of PHMB treatment on adenovirus survival at room temperature and 40°C are presented in the Table. At room temperature, PHMB at a concentration of 50 ppm produced no mean reductions of adenovirus titers greater than 1 log₁₀ for any of the adenovirus serotypes tested. At 40°C, 50 ppm of PHMB produced mean reductions in titers greater than 2 but less than 3 log₁₀ for Ad2, Ad3, Ad19, and Ad37. The mean reductions in titers for Ad1, Ad4, and Ad5 were greater than 1 but less than 2 log₁₀. The mean (SD) reduction in titer for the important ocular serotype of Ad7a approached 1 log₁₀ (−0.91 [0.26] PFU/mL), whereas 50 ppm of PHMB had no effect on Ad8 at 40°C. No virucidal reductions were demonstrated for PHMB at 50 ppm for either temperature tested.

Adenoviruses are hardy viruses that have the ability to survive at least 8 weeks in a desiccated state,¹⁷ 3 to 4 weeks in multidose bottles of topical fluorescein,¹⁸ and at least 5 days in liquid transport medium without a significant loss in infectivity after cross-country shipment.¹⁹ The hardiness of adenoviruses may contribute to their ability to cause outbreaks of ocular infections. Therefore, to prevent disease transmission, it is of utmost importance to eliminate adenoviruses from potential vectors of transmission, such as contaminated tomometer tips and ophthalmic instruments in ophthalmologists' offices and recreational water.

Many disinfectants have been tested for their abilities to kill adenoviruses. Rutala and colleagues²⁰ performed an extensive study evaluating the efficacy of a number of disinfectants against Ad8. They determined that 0.55% ortho -phthalaldehyde, 2.4% glutaraldehyde, 2.65% glutaraldehyde, 6000 ppm chlorine, 1900 ppm chlorine, 70% ethanol, 65% ethanol with 0.63% quaternary ammonium compound, 79.6% ethanol with 0.1% quaternary ammonium compound, and 0.2% peracetic acid were effective against Ad8. Other common disinfectants, such as 70% isopropyl alcohol, 3% hydrogen peroxide, 4% chlorhexidine, and 10% povidone-iodine, were ineffective against Ad8.²⁰ These results may not extend to all adenovirus types, however, because disinfectants have a range of efficacy against different adenovirus types.²¹

To our knowledge, there have been no earlier studies evaluating the efficacy of PHMB as a disinfectant against adenovirus. The results of the current study demonstrated that at room temperature, PHMB treatment at a concentration of 50 ppm produced no reductions of adenovirus titers greater than 1 log₁₀ for any of the adenovirus serotypes tested after 24 hours of incubation. We conclude from these data that PHMB is an ineffective disinfectant against adenovirus at room temperature in concentrations used in swimming pools.

At a concentration of 50 ppm, PHMB was more effective when the temperature was raised to 40°C. This concentration produced decreases in adenovirus titers greater than 1 log₁₀ for 7 of 9 adenovirus serotypes but did not demonstrate virucidal efficacy (99.9% reduction) against any of the adenovirus serotypes tested. However, 50 ppm of PHMB had little or no effect on Ad7a and Ad8, 2 major causes of ocular adenovirus infections. From these data, we conclude that PHMB may be more effective for some, but not all, adenovirus serotypes when the incubation temperature is raised to 40°C. Several factors may contribute to the increase in PHMB effectiveness at the higher temperature. Pinto et al²² demonstrated that exposure to PHMB of the MS2 bacteriophage—used as a surrogate virus for nonenveloped mammalian viruses, including adenovirus—led to the formation of viral aggregates, which were probably caused by changes in viral surface hydrophobicity. They concluded that the formation of these aggregates reduced the virucidal activity of PHMB. A follow-up study by the same group and using the same virus demonstrated that increasing the incubation temperature from 20°C to 40°C and 50°C decreased the viral aggregates and increased the antiviral activity of PHMB.²³ Pinto et al²³ speculated that high temperatures may also produce conformational changes in the viral capsid that increase the sensitivity of viral nucleocapsids to PHMB. These factors may have also contributed to an increase in effectiveness of PHMB against adenovirus at 40°C.

Based on the data from the current study, swimming pool water sanitized with PHMB would not effectively kill adenovirus contained in the water. Therefore, these swimming pools are at greater risk to act as vectors for the transmission of adenoviral ocular infections than those properly sanitized with an agent such as chlorine. Hot tub water may be more effectively sanitized with PHMB, because the increased temperature increased the effectiveness of PHMB. However, PHMB was not effective against all the adenovirus serotypes tested, so it may not be the optimal sanitizing agent for hot tub water.

These data are also important because PHMB is used as a disinfectant in several multipurpose contact lens solutions. The concentration of PHMB in these solutions is usually 0.0001% (1 ppm [μg/mL]),¹⁵ which is 50 times lower than the concentration tested in the current study. It was previously demonstrated that contact lens solutions containing 0.0001% polyaminopropyl biguanide or 3% hydrogen peroxide solution were ineffective in sterilizing contact lenses infected with Ad8 and Ad19.²⁴ On the basis of the data in the current study, we predict that the PHMB contained in multipurpose contact lens solutions would be ineffective for disinfecting contact lenses contaminated with adenovirus. Because the multipurpose contact lens solutions containing PHMB were not tested in this study, the disinfection efficacy of the formulated solutions against adenovirus remains unknown.

Correspondence: Eric G. Romanowski, MS, Eye & Ear Institute, Room 1020, 203 Lothrop St, Pittsburgh, PA 15213 (romanowskieg@upmc.edu).

Submitted for Publication: September 5, 2012; final revision received December 4, 2012; accepted December 6, 2012.

Published Online: February 28, 2013. doi:10.1001/jamaophthalmol.2013.2498

Author Contributions: Mr Romanowski confirms that he had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis, as well as the decision to submit for publication.

Funding/Support: This study was supported by the National Institutes of Health (CORE Grant for Vision Research EY08098 to the Ophthalmology and Visual Sciences Research Center and grant AI085570), the Eye and Ear Foundation of Pittsburgh, and Research to Prevent Blindness (including career development award to Dr Shanks).

Role of the Sponsors: The funding organizations had no role in the design or conduct of this research.

Previous Presentation: This study was presented in part at the 2011 Ocular Microbiology and Immunology Group annual meeting; October 21, 2011; Orlando, Florida.