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

Abstract

Purpose

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. The current study determined whether PHMB is an effective disinfectant against ocular adenovirus serotypes at a concentration used to disinfect swimming pools and hot tubs.

Methods

The direct disinfecting activity of PHMB was determined in triplicate assays by incubating nine human adenovirus types (1, 2, 3, 4, 5, 7a, 8, 19, and 37) with 50 and 0 PPM (µg/ml) of PHMB for 24 hours at room temperature, to simulate swimming pool temperatures, or 40°C, to simulate hot tub temperatures. Plaque assays determined adenovirus titers after incubation. Titers were Log₁₀ converted and mean ± standard deviation Log₁₀ reductions from controls were calculated. Virucidal (greater than 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 two adenovirus types and greater than 1 Log₁₀, but less than 3 Log₁₀, for seven of nine adenovirus types.

Conclusions

50 PPM of PHMB was not virucidal against adenovirus at temperatures consistent with swimming pools or hot tubs.

Clinical Relevance

Recreational water maintained and sanitized with PHMB has the potential to serve as a vector for the transmission of ocular adenovirus infections.

INTRODUCTION

Adenovirus (Ad) ocular infections (epidemic keratoconjunctivitis [EKC], follicular conjunctivitis [FC], and pharyngeal conjunctival fever [PCF]) are the most commonly occurring ocular viral infections in the world¹. These infections are known to occur in community and office based outbreaks¹. Outbreaks of adenoviral ocular infections can be detrimental to the community because of the significant patient morbidity and the socioeconomic consequences of lost time from school and work¹. Among the community based outbreaks, swimming pool water has been reported to be a vector for transmission^2–10. Derrick first showed in 1943 that swimming pools can act as vectors for the transmission of “swimming bath conjunctivitis”². The description of the patients’ symptoms suggested these were cases of pharyngeal conjunctival fever³. This observation came ten years before the first adenovirus was isolated¹¹. 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 for the prevention of 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 people¹². 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).

PHMB is an effective public health biocide registered for numerous applications by the U.S. EPA under the Federal Insecticide, Fungicide, and Rodenticide Act, and supported under the Biocidal Products Directive in Europe¹². It is unaffected by sunlight, water temperature, or pH fluctuations. This stability allows water to be properly maintained for longer periods, generally 7–14 days before additional PHMB is required¹³. PHMB is also used in ophthalmology as a topical treatment for Acanthamoeba keratitis¹⁴ and is an active antimicrobial agent in several multipurpose contact lens solutions¹⁵.

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

METHODS

Viruses and Cells

Clinical ocular adenovirus isolates of types 1, 2, 3, 4, 5, 7a, 8, and 19 were collected anonymously from patients presenting with typical adenoviral ocular disease at the Charles T. Campbell Ophthalmic Microbiology Laboratory at the UPMC Eye Center, University of Pittsburgh, Pittsburgh, PA and frozen at −70°C. The viruses are part of a retrospective clinical collection of adenovirus isolates. The isolates were de-identified and stored for diagnostic test validations. The serotypes of the adenovirus isolates were determined using serum neutralization. No clinical isolates of Ad37 were recovered, so the ATCC (American Type Culture Collection, Manassas, VA) reference strain of Ad37 was used. The types tested represent the most common adenovirus types that cause ocular infections (Ad8, Ad19, Ad37 cause EKC; Ad3, Ad4, Ad7a cause FC and PFC)¹ and types that can replicate in rabbit ocular models (Ad1, Ad2, Ad5)¹⁶.

A549 Human Lung Carcinoma cells (CCL-185, ATCC) were used to prepare the adenovirus stocks and for the determination of viral titers for adenovirus. The A549 cells were grown and maintained in tissue culture media containing Eagle's MEM supplemented with 10% fetal bovine serum (Sigma Cell Culture Reagents, St. Louis, MO).

Experimental Agent

Polyhexamethylene biguanide hydrochloride (Baquacil^® Sanitizer and Algistat Step 1, Arch Chemicals, Inc., Atlanta, GA) was purchased from a local swimming pool vendor in the Pittsburgh, PA area. The PHMB concentration in Baquacil^® is 20%.

In Vitro Log Reduction Virucidal Assay

This study was conducted using triplicate assays with initial inocula of approximately 1.0 × 10⁶ plaque forming units per milliliter (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 µl of each of the test viruses were added to duplicate sets of 2 ml screw capped freezing tubes (Sarstedt, Inc., Newton, NC) containing 450 µl of 55.55 PPM (µg/ml) or 0 PPM of PHMB prepared in tissue culture media. This resulted in a final PHMB concentration of 50 PPM. Fifty PPM of PHMB was used as per the manufacturer’s instruction for recreational water disinfection. One set of tubes containing the virus/PHMB mixtures was incubated for 24 hours on the 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 water bath for 24 hours to simulate hot tub conditions. After incubation, 500 µl of tissue culture media containing 20% fetal bovine serum (FBS) was added to each tube in preparation for the plaque assay.

Determination of Viral Titers (Plaque Assay)

Immediately after incubation and the addition of 500 µl of tissue culture media, the samples were serially diluted in tissue culture media containing 10% FBS 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 media containing 0.5% methylcellulose, except for Ad8 for which the wells were filled with standard tissue culture media. After 7–10 days incubation, the media was removed, the cells fixed and stained with 0.5% gentian violet solution containing formaldehyde, and the number of plaques counted using a dissecting microscope. The viral titers were calculated and expressed as PFU/ml.

Data Analysis

Viral titers were Log₁₀ converted and Log₁₀ differences in titers from the appropriate negative controls were calculated for each trial. The mean ± standard deviation Log₁₀ differences in titers were calculated from the data of the three trials. A mean reduction in titer from the negative control of at least three Log₁₀ (99.9% reduction) was considered a virucidal reduction.

RESULTS

The Effect of PHMB Treatment on Adenovirus Survival

The mean Log₁₀ reductions from three trials of PHMB treatment on adenovirus survival at room temperature and 40°C are presented in Table 1. At room temperature, 50 PPM of PHMB produced no mean reductions of adenovirus titers greater than one Log₁₀ for any of the adenovirus serotypes tested. At 40°C, 50 PPM of PHMB produced mean reductions in titers greater than two Log₁₀, but less than three Log₁₀, for Ad2, Ad3, Ad19, and Ad37. The mean reductions in titers for Ad1, Ad4, and Ad5 were greater than one Log₁₀ but less than two Log₁₀. The mean reduction in titer for the important ocular serotype of Ad7a approached a one Log₁₀ (−0.91 ± 0.26 PFU/ml) reduction while 50 PPM of PHMB had no effect on Ad8 at 40°C. No virucidal reductions were demonstrated for 50 PPM of PHMB at both temperatures tested.

Table 1.

Mean ± Standard Deviation Log₁₀ Differences in Adenoviral Titers from Control after PHMB Treatment.

Temperature	Ad1	Ad2	Ad3	Ad4	Ad5	Ad7a	Ad8	Ad19	Ad37
Room Temp	−0.69 ± 0.08	−0.77 ± 0.47	−0.37 ± 0.14	−0.05 ± 0.08	−0.72 ± 0.36	+0.89 ± 0.55	−0.27 ± 0.15	−0.15 ± 0.14	−0.23 ± 0.19
40°C	−1.30 ± 1.07	−2.65 ± 0.23	−2.36 ± 0.23	−1.44 ± 0.40	−1.57 ± 0.66	−0.91 ± 0.26	+0.13 ± 0.27	−2.49 ± 1.47	−2.60 ± 0.38

COMMENT

Adenoviruses are hardy viruses that have the ability to survive at least 8 weeks in a desiccated state¹⁷, 3–4 weeks in multi-dose bottles of topical fluorescein¹⁸, and at least five days in liquid transport media without a significant loss of infectivity after cross-country shipment¹⁹. The hardiness of adenoviruses may contribute to their ability to cause outbreaks of ocular infections. Therefore the disinfection of adenoviruses from potential vectors of transmission such as contaminated tomometer tips and ophthalmic instruments in the ophthalmologists’ offices and recreational water is of utmost importance to prevent transmission of disease.

Many disinfectants have been tested for their abilities to kill adenoviruses. Rutala and colleagues performed an extensive study evaluating the efficacy of a number of disinfectants against Ad8²⁰. They determined that 0.55% ortho-phthalaldehyde, 2.4% glutaraldehyde, 2.65% glutaraldehyde, 6,000 PPM chlorine, 1,900 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 Ad8²⁰. However other common disinfectants such as 70% isopropyl alcohol, 3% hydrogen peroxide, 4% chlorhexidine, and 10% povidone-iodine were ineffective against Ad8²⁰. However, these results may not extend to all adenovirus types since it is known that disinfectants have a range of efficacy against different adenovirus types²¹.

To date, there have been no studies evaluating the efficacy of PHMB for the disinfection of adenovirus. The results of this study demonstrated that at room temperature, 50 PPM of PHMB treatment produced no reductions of adenovirus titers greater than 1 Log₁₀ for any of the adenovirus serotypes tested after 24 hours incubation. We conclude from these data that PHMB is an ineffective disinfectant for adenovirus at room temperature in concentrations used in swimming pools.

Fifty PPM of PHMB was more effective when the temperature was raised to 40°C. Fifty PPM of PHMB 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, which are two 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 al. demonstrated that exposure of the MS2 bacteriophage, used as a surrogate virus for non-enveloped mammalian viruses of which adenovirus is one, to PHMB resulted in the formation of viral aggregates. The aggregates were likely caused by changes in viral surface hydrophobicity. They concluded that the formation of these aggregates reduced the virucidal activity of PHMB²². A follow-up study by the same group and using the same virus demonstrated that by increasing the incubation temperature from 20°C to 40°C and 50°C, the viral aggregates decreased and the antiviral activity of PHMB increased²³. These authors speculated that high temperatures may also produce conformational changes in the viral capsid that increase the sensitivity of viral nucleocapsids to PHMB²³. 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 a 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 therefore 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 µg/ml [PPM])¹⁵, which is 50× lower than the concentration tested in the current study. We 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 Ad19²⁴. Based on the data in the current study, we predict the PHMB contained in multipurpose contact lens solutions would be ineffective for the disinfection of contact lenses contaminated with adenovirus. As the multipurpose contact lens solutions containing PHMB were not tested in this study, the disinfection efficacy of the formulated solutions against adenovirus remains unknown.