Antiviral activity of contact lens care solutions and rub-and-rinse regimen against adenovirus

Human adenoviruses (HAdVs) are nonenveloped, double-stranded DNA viruses.¹ They are highly resistant to environmental desiccation, remaining infectious for many days on different surfaces, including plastic or metal, and eye drop bottles.^2–4 Moreover, as nonenveloped viruses, HAdVs are resistant to multiple disinfectants due to the physical properties of their capsid proteins.⁴ Therefore, they are capable of both direct and indirect transmissions of a wide spectrum of infectious diseases.^1,5,6 Human adenoviruses are divided phylogenetically into seven species (A to G): HAdV-B, HAdV-C, and HAdV-E are mainly linked with respiratory disease, HAdV-A, HAdV-D, HAdV-F, and HAdV-G with gastrointestinal disease, and HAdV-D and HAdV-E with ocular conditions.⁷

Human adenoviruses are the leading cause of acute viral conjunctivitis, accounting for up to 75% of all conjunctivitis cases.⁸ The most common ocular presentations are epidemic keratoconjunctivitis, pharyngoconjunctival fever, and isolated follicular conjunctivitis.^8,9 Epidemic keratoconjunctivitis is a highly contagious and potentially vision-disabling infection of the conjunctiva and cornea.⁶ It is associated with nosocomial outbreaks and outbreaks in highly populated settings, such as schools, nursing homes, workplaces, and swimming pools.^9–12 Among various serotypes of adenovirus that have been isolated, at least 19 can cause keratoconjunctivitis. Serotypes 8, 19, and 37 are the most frequent serotypes associated with keratoconjunctivitis. Although less frequent, serotype 5 has also been associated with keratoconjunctivitis and pharyngoconjunctival fever as well.^13,14 The effects of HAdv on the cornea and vision can be significant, including infiltrates and light sensitivity, and there exists a risk of transmission via multiuse contact lenses.^15,16

It is estimated that more than 140 million people worldwide use contact lenses for vision correction.¹⁷ To minimize ocular infections transmitted through keratoconjunctivitis, reusable contact lenses must be cleaned and disinfected daily using an approved contact lens care product.¹⁸ Regulatory approval of those products requires demonstration of their antimicrobial efficacy against standard bacteria and fungi strains by fulfillment of the minimum requirements defined by International Organization for Standardization (ISO) 14729.¹⁹ Interestingly, requirements regarding the efficacy of contact lens care products against viruses have not yet been established. There are two main types of disinfectant systems, oxidative and nonoxidative, available in the market.

Nonoxidative systems utilize complex formulations that contain several chemical components including biocides, such as polyhexamethylene biguanide, polyaminopropyl biguanide, myristamidopropyl dimethylamine (Aldox), and polyquaternium, as well as various surfactants, wetting, chelating, and buffering agents.¹⁸ The simplicity, convenience, and low cost of nonoxidative systems make them a popular choice to be prescribed by practitioners and used by patients. All contact lens care products based on nonoxidative systems require a rub-and-rinse step prior to disinfection, and therefore, patient compliance with manufacturer instructions is crucial for their maximal efficacy. Several studies have shown the importance of rub and rinse to remove bacterial, fungal, and protozoan contamination from reusable contact lens materials.^20–23 Although very few studies are available evaluating nonoxidative contact lens care products against viruses, the efficacy of rub-and-rinse steps on removing enveloped²⁴ as well as nonenveloped viruses, including adenovirus type 8, from contact lens surfaces has been highlighted.²⁵

Among the oxidative contact lens care products, those based on 3% hydrogen peroxide are the most widely used. In Japan, Europe, New Zealand, and Hong Kong, products containing povidone-iodine (PVP-I) are also available.^26–32 All lenses exposed to these two oxidative care systems require neutralization of the oxidizing agent before reapplication onto the eye. In so-called one-step systems, disinfection and neutralization occur simultaneously. In the most commonly used hydrogen peroxide systems, the peroxide is neutralized by a platinum disc present in the case supplied with the product, whereas in the PVP-I systems, the neutralization is achieved by adding a tablet containing a neutralizing agent such as ascorbic acid.²⁹ Although limited data are available regarding the efficacy of these solutions against viruses, both hydrogen peroxide and PVP-I have shown higher disinfection efficacy against enveloped viruses than nonoxidative-based systems.^33,34 To date, only one published study has evaluated an oxidative contact lens care product against adenoviruses, using a suspension test. Kowalski and colleagues³⁵ demonstrated that, although hydrogen peroxide significantly reduced the level of adenovirus contamination on contact lens surfaces, infectious viral particles were still recovered after the recommended disinfection time. Therefore, the authors recommended that patients with adenoviral eye infections dispose of their contaminated contact lenses, as it could still serve as a reservoir for adenovirus spread to the community even after being processed with a care system.³⁵

Considering the clinical relevance of eye infections caused by HAdVs and the lack of data surrounding the efficacy of contact lens care products against these viruses, this study was designed to evaluate the efficacy of nonoxidative and oxidative contact lens care products against adenovirus type 5 (Ad5). Adenovirus type 5 is a well-characterized strain that has been used extensively to introduce genetic material into host cells, and it can be easily amplified at a high titer.

First, a modified suspension test from ISO 14729 “Ophthalmic Optics—Contact Lens Care Products—Microbiological Requirements and Test Methods for Products and Regimens for Hygienic Management of Contact Lenses” was used to individually test the investigated contact lens care products against adenovirus preparations, in the absence of contact lenses. Then, the influence of a variety of hard and soft lens materials on adenovirus attachment and the influence of a rub-and-rinse step to remove these viruses from the contact lens surfaces were evaluated. Additionally, the influence of compliance with specific manufacturer instructions for using the investigated systems to remove adenoviral contamination from two representative soft lens materials (etafilcon A and lotrafilcon B) was also evaluated.

METHODS

This study received ethics approval from the Office of Research Ethics at the University of Waterloo under application number 43217: Interaction of viruses with contact lenses, spectacles, and contact lens solutions.

Contact lenses care products

The contact lens care products included in this study were Biotrue (Bausch & Lomb, Rochester, NY), Boston Simplus (Bausch & Lomb), OPTI-FREE Puremoist (Alcon, Fort Worth, TX), Clear Care (Alcon), cleadew (Ophtecs, Kobe, Japan), and cleadew GP (Ophtecs) (Table 1). The contact lens case supplied with Clear Care and the tablets supplied with cleadew and cleadew GP were used according to the manufacturer’s instructions to ensure that neutralization occurred during the appropriate disinfection time. Clear Care cases contain a platinum disc that gradually reduces the hydrogen peroxide to water and oxygen over 6 hours of incubation. Ascorbic acid and sodium sulfite, present in both cleadew and cleadew GP tablets, neutralize the PVP-I to a safe level over 4 hours of incubation. Moreover, the solution color change from orange to colorless indicates that the PVP-I neutralization has occurred appropriately.³⁴

Virus and cell line

Human Ad5 (VR-5), a strain originally isolated from an adenoid tissue, was propagated in HEK-293T cells (human kidney epithelial cell; American Type Culture Collection CRL-3116), which were maintained in Dulbecco modified Eagle medium (DMEM) (Wisent BioProducts, Saint-Jean-Baptiste, Quebec, Canada) supplemented with 10% fetal bovine serum in a 5% CO₂ atmosphere at 37°C and 100% humidity.

Virus titration

The end-point dilution assay was used to measure the viral load of Ad5 using HEK-293T cells. It was conducted as per the Tissue Culture Infectious Dose 50 (TCID50) assay,³⁶ which is defined as the dilution of a virus required to infect 50% of a given cell culture, and analyzed using the Most Probable Number calculator.³⁷ Briefly, 10-fold serial dilutions of samples were added to 96-well plates with seeded HEK-293T cells at a density of 5 × 10⁵ cells/mL. An extra row of mock-infected cells was included as a control. The plates were incubated at 37°C and 5% CO₂ for 10 days. Then, each well was examined using light microscopy (Zeiss Axiovert 40C, Göttingen, Germany) and scored as infected or noninfected based on cytopathic effects. As reported by American Type Culture Collection, cytopathic effect of HEK293 cells is characterized by presence of morphological cell changes that can be easily identified under light microscopy, which includes cell clustering, rounding, and sloughing. In all experiments, cell morphology was always carefully compared with the control, in which HEK-293 cells were not infected. The log reduction of virus titer was calculated as the difference between the virus titer after contact with contact lens solutions and the control.

Cytotoxicity and neutralization controls

Cytotoxicity and neutralization studies with Ad5 and contact lens care products were performed as previously described.³⁴ For cytotoxicity tests, 99 parts of the disinfecting solutions were mixed with 1 part of DMEM + 2% fetal bovine serum instead of Ad5 and incubated for 4 and 6 hours at room temperature. Samples were then subjected to the same procedures to determine the virus titer (Methods – Virus titration). After incubation of the plates, cytopathic effect at each dilution was observed to determine the lowest contact lens solution concentration at which cytotoxic effects were absent. For neutralization tests, the remaining volume of cytotoxicity tests was diluted at 1:10 with cold cell culture medium, except for cleadew GP, which was diluted at 1:100 (to avoid cytotoxic effects on host cells—see Results – Cytotoxicity and neutralization controls). Then, 99 parts of those dilutions were mixed with 1 part of Ad5 suspension and incubated for 4 and 6 hours at room temperature. The virus titer was determined as described in Methods – Virus titration. For test validation, the difference between negative control (Dulbecco phosphate-buffered saline [D-PBS], pH 7.4) and neutralization controls should be ≤0.5 log₁₀ steps.³⁸

Quantitative suspension test

As ISO 14729 recommends that the volume of inoculum does not exceed 1% of the sample volume on stand-alone tests, 99 parts of each contact lens product were mixed with 1 part of Ad5 (10⁸ MPN/mL).¹⁹ As a control, 99 parts of D-PBS were mixed with 1 part of Ad5. All mixtures were incubated for both 4 and 6 hours at room temperature. Then, the virucidal activity of the contact lens care product was immediately suppressed by a dilution at 1:10 with cold DMEM, except for cleadew GP, which was diluted at 1:100 (see Results – Cytotoxicity and neutralization controls). All samples were then serially diluted to determine the virus titer via TCID50 end-point dilution assay (Methods – Virus titration).³⁶ All tests were run in triplicates.

Time-point quantitative suspension test for Clear Care

For Clear Care, an additional stand-alone test was performed to analyze if an increased incubation period before neutralization could increase its virucidal activity. In the Clear Care case, 99 parts of the solution were mixed with 1 part of Ad5 (10⁸ MPN/mL), but the lid of the case containing the platinum disk responsible for the hydrogen peroxide neutralization was not added. After 0, 2, 4, and 6 hours, the case was then closed with its lid, starting the 6-hour incubation period recommended by the manufacturer. The time point 0 hour indicates that the contact lens case was immediately closed with its lid after mixing the contact lens solutions with Ad5. Another time point was also included, in which 99 parts of Clear Care solution were mixed with 1 part of Ad5 (10⁸ MPN/mL); the contact lens case was immediately closed with its lid and then incubated for 12 hours instead of 6 hours as recommended by the manufacturer. For each time point, a control was included, in which 99 parts of D-PBS instead of Clear Care solution were mixed with 1 part of Ad5 (10⁸ MPN/mL).

Impact of rub-and-rinse regimen on Ad5 removal from contact lens surfaces using D-PBS

The Ad5 binding rates to contact lenses and the impact of rub and rinse at removing virus from their surfaces were evaluated as described in a previous publication.²⁴ All lenses included in this experiment were tested in triplicate using three of each contact lenses material (Table 2). The day before each experiment, contact lenses were removed from their packaging and soaked in D-PBS overnight. Then, lenses were individually soaked in 4 mL of Ad5 (10⁶ MPN/mL) and incubated for 6 hours at room temperature. As a control, the Ad5 suspension was incubated in the same condition, but with no contact lenses. Thereafter, lenses were submitted to four different treatments (Fig. 1). Rinses were performed in 6-well plates. In the first condition, termed “no rub and rinse,” lenses were removed from the virus suspension and briefly dipped in 4 mL of D-PBS. In the second condition, termed “rinse,” each lens was transferred to a new well, and one side of the lens was rinsed with 4 mL of D-PBS. Then, each lens was transferred to a new well, and the other side of the lens was also rinsed with 4 mL of D-PBS (no rub was performed). In the third condition, termed “rinse twice,” each side of the lens was rinsed twice with 4 mL of D-PBS (no rub was performed). Each rinse was performed as described above. In the fourth and last condition, termed “rub and rinse,” each side of the lens was rinsed with 4 mL of D-PBS and rubbed in a circular motion (5×) using a nitrile-gloved 3D-printed “finger” of physiological proportions, and then each side of the lens was rinsed again with 4 mL of D-PBS. After these treatments, contact lenses were immediately vortexed in 1 mL of D-PBS, and then infectious virus particles were quantified as described in Methods – Virus titration.

Impact of rub and rinse on Ad5 removal from contact lens surfaces using contact lens care products

As in the previous experiments, lenses were removed from their blister packs and soaked in D-PBS overnight. Then, the lenses were individually soaked in 4 mL of Ad5 suspension (10⁶ MPN/mL) and incubated for 6 hours at room temperature. In these experiments, two soft contact lens materials, one conventional hydrogel (etafilcon A) and one silicone hydrogel (lotrafilcon B), were selected to be disinfected using two contact lens care products based on nonoxidative disinfecting systems (Biotrue and OPTI-FREE Puremoist) and two based on oxidative disinfecting systems (Clear Care and cleadew). For both etafilcon A and lotrafilcon B, two sets of three lenses were disinfected using each contact lenses care product included in this experiment. The first set of lenses was disinfected following all the manufacturers’ recommendations. However, in the second set of lenses, rub-and/or-rinse steps were not performed as recommended by the manufacturers to garner their impact on the recommended care actions. For either Biotrue or OPTI-FREE Puremoist, rub-and-rinse steps prior to the disinfection, which are required, were not included; for Clear Care, the required rinsing step prior to the 6-hour disinfection was not performed, and for cleadew, a rinse step after the 4-hour incubation was not included (Table 3). After disinfection, all contact lenses were individually vortexed in 1 mL of D-PBS, and infectious viral particles were determined as described in Methods – Virus titration.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism version 9.2.0 (GraphPad Software, Boston, MA, USA). Infectious virus particles are expressed as mean and standard deviation. The difference in mean infectious virus particles between test conditions was tested using unpaired t test or one-way analysis of variance with Tukey post hoc multiple comparisons test. The values of infectious virus particles below the limit of quantification were assigned a “0.” A p value of 0.05 or below was considered statistically significant.

RESULTS

Cytotoxicity and neutralization controls

Prior to performing experiments involving the various care products, both cytotoxicity and neutralization controls were run. The cytotoxicity controls revealed that Biotrue, Boston Simplus, OPTI-FREE Puremoist, and cleadew GP were cytotoxic for HEK-293T cells. A 1:10 dilution with cold DMEM was efficient to avoid cytotoxic effects of Biotrue, Boston Simplus, and OPTI-FREE Puremoist to the host cell line, whereas 1:100 dilution was needed to avoid those effects caused by cleadew GP. Clear Care and cleadew did not show cytotoxic effects on HEK-293T cells. However, to obtain the same experimental conditions for all products tested, samples containing Biotrue, Boston Simplus, OPTI-FREE Puremoist, and cleadew were diluted 1:10 before the performance of TCID50 assay, whereas samples containing cleadew GP were diluted 1:100. Due to these additional dilutions, the limit of quantification of TCID50 assay was 7.9 × 10¹ MPN/mL for all contact lens care products, except for cleadew GP, which was 7.9 × 10² MPN/mL. The results of neutralization controls also confirmed that all contact lens care products were efficiently neutralized after the samples were diluted 1:10 and 1:100, because the difference in virus titers between all neutralization controls and the negative control was lower than ≤0.5 log₁₀ (Appendix Table A1, available at https://links.lww.com/OPX/A794).

Quantitative suspension tests

Nonoxidative disinfecting systems

Biotrue and OPTI-FREE Puremoist did not exhibit a significant log₁₀ reduction compared with the controls after 4-hour incubation (p>0.05) (Fig. 2). However, both Biotrue and OPTI-FREE Puremoist exhibited a significant log₁₀ reduction of Ad5 compared with the control after 6-hour incubation, showing 0.42 (±0.13) and 0.42 (±0.15) log₁₀ reduction, respectively (all p<0.01), indicating that it was able to inactivate 31.8% (±11.5%) and 29.9% (±11.4%) of the infectious virus. Boston Simplus also exhibited a significant log₁₀ reduction of Ad5 compared with the control after either 4 or 6 hours, showing 0.49 (±0.17) and 0.76 (±0.14) log₁₀ reduction, respectively (all p<0.01), indicating that it was able to inactivate 65.0% (±14.57%) and 82.0% (±5.7%) of the infectious virus (Appendix Table A2, available at https://links.lww.com/OPX/A794).

Oxidative disinfecting systems

Clear Care did not exhibit a significant log₁₀ reduction compared with the controls after 4-hour incubation (p>0.05) Fig. 2. However, it exhibited a significant log₁₀ reduction of Ad5 compared with the control after 6-hour incubation, showing 0.52 (±0.09) log₁₀ reduction (p<0.01), indicating that it was able to inactivate 69.5% (±6.5%) of the infectious virus at this time point. Moreover, increasing the contact time of Ad5 and 3% hydrogen peroxide before its neutralization led to an increased log₁₀ reduction, showing 1.80 (±0.10), 2.15 (±0.09), and 2.18 (±0.08) log₁₀ reduction after 2, 4, and 6 hours of exposure prior to neutralization, respectively, indicating that it was able to inactivate 98.5% (±0.4%), 99.3% (±0.1%), and 99.3% (±0.2%) (Fig. 3 and Appendix Table A3, available at https://links.lww.com/OPX/A794) of the infectious virus if not immediately neutralized by the platinum disc. In contrast, the oxidative disinfecting systems based on 0.05% PVP-I (cleadew and cleadew GP) showed more than 3 log₁₀ reduction compared with the controls after both 4- and 6-hour incubation (all p<0.01). cleadew showed 4.48 (±1.12) and 5.30 (±1.04) log₁₀ reduction after 4 and 6 hours, respectively, indicating that it was able to inactivate 99.992% (±0.009%) and 99.999% (±0.002%) of Ad5 after these time points. cleadew GP showed 3.63 (±0.22) and 4.04 (±0.15) log₁₀ reduction after 4 and 6 hours, respectively, indicating that it was able to inactivate 99.988% (±0.006%) and 99.997% (±0.001%) (Appendix Table A2, available at https://links.lww.com/OPX/A794).

Effect of rub and rinse on Ad5 removal from contact lens surfaces using D-PBS

As observed in Fig. 4, after “no rub or rinse” treatment, approximately 10² to 10³ infectious viral particles were recovered from each contact lens tested, lowering viral titers by 2.40 to 3.50 log₁₀ compared with the control. When the “rinse” treatment was performed, approximately 10 to 100 infectious viral particles were recovered, indicating a viral titer reduction of 4.20 to 5.40 log₁₀ compared with control. For lenses submitted to the “rinse twice” treatment, the average of infectious viral particles recovered was below the limit of quantification for most of the lenses tested, except for omafilcon A and fluoroxyfocon HD, because in one or two replicates, no virus was detected, whereas in the other replicates, virus was detected around the limit of quantification. This treatment lowered viral titers by more than 4.93 log₁₀ compared with the control. However, when lenses were submitted to the “rub and rinse” treatment, no infectious viral particles were recovered, indicating that the virus titer was reduced to below the limit of quantification for all lenses tested.

Efficacy of contact lens care products to remove Ad5 from contact lens surfaces

Because only a simple rinse step removed Ad5 from all materials, only two representative soft lens materials (one conventional hydrogel and one silicone hydrogel) underwent exposure to the care systems to determine their ability to remove the bound virus particles. The nonoxidative disinfection systems (Biotrue and OPTI-FREE Puremoist) were able to reduce the number of infectious viral particles for both etafilcon A and lotrafilcon B to below the limit of quantification only when rub-and-rinse steps were included as recommended by the manufacturers. When rubbing and rising were not performed, averages of 9.08 × 10² (SD, 9.37 × 10²) and 2.90 × 10³ (SD, 6.83 × 10²) infectious viral particles/lens were recovered from lotrafilcon B when lenses were incubated in Biotrue and OPTI-FREE Puremoist (both p=0.01), respectively. Similarly, 3.04 × 10³ (SD, 4.15 × 10²) and 2.22 × 10³ (SD, 3.32 × 10²) infectious viral particles/lens were recovered from etafilcon A when lenses were incubated in Biotrue and OPTI-FREE Puremoist (both p<0.01), respectively, without rubbing and rinsing (Fig. 5). Among the tested contact lens care products based on an oxidative disinfection system, cleadew was able to reduce the number of infectious viral particles that adhered to both etafilcon A and lotrafilcon B to below the limit of quantification, even when the lenses were not rinsed with the contact lens solution after the incubation period, as recommended by the manufacturers. However, infectious viral particles were still recovered when both lotrafilcon B and etafilcon A were exposed to Clear Care. Averages of 2.69 × 10² (SD, 7.45 × 10¹) and 2.89 × 10² (SD, 1.10 × 10²) infectious viral particles/lens were recovered from lotrafilcon B and etafilcon A, respectively, when lenses were simply soaked in Clear Care, and averages of 1.65 × 10² (SD, 5.38 × 10¹) and 1.48 × 10² (SD, 5.37 × 10¹) infectious viral particles/lens were recovered from lotrafilcon B (p=0.01) and etafilcon A (p<0.01), respectively, when disinfecting using the manufacturer’s recommendations (Fig. 5).

DISCUSSION

In the first part of this study, the disinfecting efficacy of six commercially available contact lens care products against adenovirus was successfully examined using a quantitative suspension test. An Ad5 stock was mixed with each test contact lens care product, and the mixture was then incubated for 4 or 6 hours. At the end of the product-virus contact time, the test product was neutralized, and then the number of infectious viral particles was determined using the TCID50 assay.

The efficacy and validity of these assays for adenovirus work were performed in several ways, which was particularly important considering that standardized methods of virus neutralization with contact lens care products have not been defined. First, the toxicity of the contact lens care products to the host cells used in the TCID50 assay was evaluated by performing the cytotoxicity control (Methods – Cytotoxicity and neutralization controls). Because no virus is added in this experiment, any morphological change or cell damage indicates that the product is cytotoxic to the cells. In this study, Biotrue, Boston Simplus, OPTI-FREE Puremoist, and cleadew GP caused cytotoxic effects on HEK-293T cells. This could lead to false-positive results, because it could be mistaken to be cytopathic effects caused by virus infection. According to the American Society for Testing and Materials International Standard E1052-20,³⁹ reduction in cytotoxicity can be achieved by different methods, including additional dilution with cell culture medium. Therefore, it was verified that an additional 1:10 dilution with cold DMEM immediately after the incubation period was efficient to avoid those cytotoxic effects caused by Biotrue, Boston Simplus, and OPTI-FREE Puremoist, whereas 1:100 dilution was needed to avoid cleadew GP cytotoxicity effects on HEK-293T cells.

Following the validation of cytotoxicity controls, the neutralization controls were performed to ensure that all products were efficiently neutralized at the end of the care product–virus contact time. The test product neutralization is extremely important to avoid an extension of the incubation period, because the residual product could inhibit virus propagation and lead to false-positive results. According to the American Society for Testing and Materials International Standard E1052-20,³⁹ neutralization can be achieved using a chemical neutralizer agent or performing a dilution. Clear Care, cleadew, and cleadew GP, all products based on oxidative systems, already have a neutralizing agent in their formulation. The case supplied with Clear Care contains a platinum disk that fully neutralizes the hydrogen peroxide to water and oxygen after a 6-hour incubation period. The tablets supplied with cleadew and cleadew GP contain ascorbic acid and sodium sulfite, respectively, which completely neutralize the PVP-I present in both products after a 4-hour incubation period. However, Biotrue, Boston Simplus, and OPTI-FREE Puremoist, products based on nonoxidative systems, do not contain a neutralizer agent in their systems. After both the incubation period of the contact lens care products and adenovirus and the performance of the 1:10 and 1:100 additional dilutions (to avoid toxic effects on host cell lines, as explained above), it was expected that all contact lens care products were neutralized by the neutralizer present in their formulations or by those additional dilutions. In fact, all products were efficiently neutralized, because the difference in virus titers between all neutralization controls and the negative control was lower than ≤0.5 log₁₀ (Appendix Table A1, available at https://links.lww.com/OPX/A794).

The results of both cytotoxicity and neutralization controls confirmed that all care products were efficiently neutralized and nontoxic to the cells and virus after the product-virus contact time, validating the protocols, which were subsequently used to evaluate the ability of care products in reducing the viral load of Ad5.

It was found that infectious viral particles of Ad5 were still recovered when directly mixed and incubated with nonoxidative systems. Previous studies have considered a product as effective against viruses when it provides 3 to 4 log₁₀ reductions in the number of infectious viral particles.^25,33,40 Biotrue, Boston Simplus, and OPTI-FREE Puremoist did not provide more than 3 log₁₀ reductions of infectious viral particles compared with the control, and therefore, they cannot be considered effective against adenovirus based on the components within the solutions alone. Other studies have also demonstrated that care products based on nonoxidative system have minimal ability to inactivate both enveloped viruses, such as human coronaviruses,³⁴ murine coronavirus,³³ and herpes simplex virus type 1,²⁵ and nonenveloped viruses, such as adenovirus type 8 and poliovirus type 2.²⁵ In all those studies, the test products based on nonoxidative system also showed less than 3 log₁₀ reduction of infectious viral particles compared with the control in quantitative suspension assays.

For the oxidative systems, in this study, Clear Care also did not provide more than 3 log₁₀ reductions compared with the control, even when the product-virus contact time was increased by 6 hours before neutralization. In general, hydrogen peroxide is considered an efficient disinfectant showing bactericidal, virucidal, sporicidal, and fungicidal properties.⁴¹ However, to date, there is a lack of studies evaluating 3% hydrogen peroxide solution as a simple germicide against adenovirus using quantitative suspension tests. Because surface contamination plays an important role in the transmission of several viruses, including adenoviruses, most of the studies available evaluated the efficacy of different methods using hydrogen peroxide to disinfect contaminated surfaces and medical materials.^40,42–44 Rutala and colleagues,⁴⁰ for example, demonstrated the inefficacy of 3% hydrogen peroxide solution to inactivate adenovirus type 8 on stainless steel surfaces. Because other recent studies have demonstrated the ineffectiveness of 3% hydrogen peroxide against adenovirus, the Centers for Disease Control and Prevention no longer recommends the disinfection of tonometers with 3% hydrogen peroxide because it has been associated with adenovirus keratoconjunctivitis outbreaks.⁴⁵ Conversely, studies have shown that contact lens care products containing 3% hydrogen peroxide are often effective against enveloped viruses. In our previous publication, Clear Care reduced the number of infectious viral particles of human coronaviruses to below the limit of quantification.³⁴ Yasir and colleagues³³ showed that AOSept Plus (Alcon), another 3% hydrogen peroxide solution, also reduced the number of infectious murine coronavirus to below the limit of quantification. It is well known that enveloped viruses are more susceptible than nonenveloped viruses.⁴⁶ The first has a host-derived membrane, called a viral envelope, which is required for infection. The envelope is composed mainly of a lipid bilayer, which is associated with their relatively low resistance to desiccation, heat, alcohols, or detergents.^47,48 In contrast, nonenveloped viruses utilize a protein coat for infection, and therefore, inactivation often requires denaturation of the viral capsid proteins or essential replicative proteins.⁴⁹ Hydrogen peroxide produces highly reactive hydroxyl free radicals that can cleave or crosslink a large range of essential biomolecules, including lipids, such as those present in the virus envelope, leading to virus inactivation.⁵⁰ Interestingly, although using different concentrations and virus-contact time, some studies have shown that hydrogen peroxide is efficient against some nonenveloped viruses, such as poliovirus⁵¹ and norovirus.^52,53 In fact, the reactive hydroxyl free radicals can also inactivate proteins, such as those present in the viral capsid of nonenveloped viruses. However, studies have shown that differences in the virucidal activity of chemical biocides against nonenveloped viruses depend on the composition of the viral capsid, as well as on the virus hydrophilicity and lipophilicity, which could explain the adenovirus resistance to hydrogen peroxide demonstrated in the current study.⁵⁴

The PVP-I–based oxidative systems, cleadew and cleadew GP, showed a significantly higher disinfection efficacy than the other care products evaluated. Both cleadew and cleadew GP showed more than 3 log₁₀ reduction compared with the controls after both 4- and 6-hour incubation, inactivating more than 99.9% of viral particles. Povidone-iodine has been reported as a broad-spectrum microbicide solution against bacteria, fungi, protozoans, and several viruses for more than a century.^8,55 Since its discovery, PVP-I has been extensively used in the medical field as an antiseptic and disinfectant agent in laboratories and health care facilities.⁸ The PVP-I consists of elementary iodine bound to the carrier poly(1-vinyl-2-pyrrolidone), which releases free iodine that reacts with cell membrane proteins as well as with mitochondrial enzymes, leading to microorganism inactivation.⁵⁶ A systematic review demonstrated that the best virucidal efficacy is achieved when PVP-I is used in a concentration range of 0.08 to 0.9%, with a maximum tested exposure time of 60 minutes.⁵⁴ Additionally, studies have also demonstrated that the sensitivity of adenovirus serotypes to PVP-I differs markedly.^57,58 Both cleadew and cleadew GP contain 0.05% PVP-I, and a few studies are available evaluating concentrations of 0.009 to 0.05% PVP-I against adenovirus, specifically the serotype 5 used in this study.^57–59 Kawana and colleagues⁵⁹ demonstrated that Ad5 was inactivated for more than 3 log₁₀ only when doses higher than 0.5% PVP-I were tested. For virucidal testing, they used equal volumes of PVP-I solution and virus suspension, whereas only 1% of inoculum was used in this study. Moreover, the contact time tested was only 60 seconds, whereas 4- and 6-hour incubation periods were tested in this study.⁵⁹ Sauerbrei and colleagues⁵⁷ evaluated the virucidal activity of a liposomal PVP-I against several adenovirus serotypes. They demonstrated that 0.05% PVP-I provided less than 3 log₁₀ reduction of Ad5; however, higher inoculum concentrations (virus titer ranges of 10⁷ to 10¹¹ TCID50/mL) and shorter contact times (0.5 to 60 minutes) were used, compared with this study.⁵⁷ The variety of conditions used in those previous publications, mainly the use of both higher virus stock titers and higher percentages of inoculum, as well as the shorter exposure contact time, could explain the poor effectiveness of 0.05% PVP-I against Ad5.

In the second part of this study, contact lenses were contaminated with Ad5 and then submitted to four different treatments to evaluate the influence of a rub-and-rinse step to remove adenovirus from a variety of contact lens materials, including conventional and silicone hydrogel, and rigid lenses. Only D-PBS (saline) was used in all treatments performed in these experiments, and no care products were used to demonstrate the impact of physical cleaning on Ad5 removal. After lenses were contaminated with adenovirus, lenses were first soaked in D-PBS alone with no rub-and-rinse steps, resulting in 10² to 10³ infectious viral particles being recovered from all lens materials. The performance of a single rinse on each side of the lens with D-PBS significantly reduced the recovery of infectious viral particles to 10¹ to 10,² whereas the performance of two rinses on each side of the lens reduced the recovery to below the limit of quantification, except for the omafilcon A and fluoroxyfocon A with Hydra-PEG materials. The material properties of those lenses could be related to these results, but further analyses are necessary to correlate those lens materials with higher virus adhesion. Although adenovirus was still recovered from omafilcon A and fluoroxyfocon A with Hydra-PEG, the number of infectious viral particles was significantly lower compared with those lenses soaked only into D-PBS. When lenses were rubbed and rinsed with D-PBS, no infectious viral particles were recovered from all tested materials. These results indicate that adenovirus binds very loosely to the contact lens materials, and the virus can be easily removed when rubbed or rinsed only with D-PBS. To date, only a few studies are available evaluating rub-and-rinse steps to remove viruses from contact lens surfaces. In our previous publication, only a single rinse step with D-PBS reduced infectious human coronavirus particles to below the limit of quantification for all tested materials,²⁴ whereas in this study, two rinses or a combined rub-and-rinse step was needed to eliminate adenovirus particles to below the limit of quantification. This difference among the tested viruses could be due to the titer of the virus suspension initially used to contaminate the lenses. As the concentration of adenovirus suspension was higher (10⁶ MPN/mL) than the human coronavirus (10⁵ MPN/mL), more disinfection steps were likely needed to reduce the adenovirus contamination. This difference could also be due to the virus structures differences, as adenovirus is a nonenveloped virus, whereas coronavirus is an enveloped virus.

As contamination with adenovirus was easily reduced on all 12 tested materials using a simple cleaning regimen with D-PBS, only one conventional hydrogel (etafilcon A) and one silicone hydrogel (lotrafilcon B) material were selected to test the efficacy of Biotrue, OPTI-FREE Puremoist, Clear Care, and cleadew on Ad5 removal. Although the nonoxidative disinfection systems did not exhibit significant efficacy against Ad5 in the quantitative suspension assays, Biotrue and OPTI-FREE Puremoist reduced the number of infectious adenovirus particles for both etafilcon A and lotrafilcon B to below the limit of quantification when rub-and-rinse steps were included as recommended by the manufacturers after Ad5 contamination. Previous studies have also demonstrated the importance of a rub-and-rinse step to reduce the numbers of viral particles adhered to contact lenses when a nonoxidative disinfection system is used. In our previous work, Biotrue and OPTI-FREE were able to reduce the number of infectious human coronavirus (HCoV-229E) adhered to etafilcon A and lotrafilcon A to below the limit of quantification only when lenses were rubbed and rinsed as recommended by the manufacturers.²⁴ Similarly, Yasir and colleagues³³ showed the efficacy of Biotrue to reduce the number of murine coronaviruses adhered to etafilcon A using a rub-and-rinse regimen. Heaselgrave and colleagues²⁵ demonstrated the ability of MeniCare Soft Multipurpose Solution (Menicon, Nagoya, Japan), which contains the same disinfectant agent found in Biotrue (0.0001% polyaminopropyl biguanide), to reduce the number of herpes simplex virus (type 1), adenovirus (type 8), and poliovirus adherent to contact lenses only when a rub-and-rinse regimen was used. Similarly, another product based on a nonoxidative disinfection system, ReNu Multipurpose (Bausch & Lomb), which contains 0.00005% polyaminopropyl biguanide, significantly reduced the number of HIV on contact lenses when a rubbing step was performed.⁶⁰ Regarding the care products based on oxidative systems, adenovirus particles were still recovered when both lotrafilcon B and etafilcon A were disinfected with the hydrogen peroxide–based Clear Care. The manufacturer recommends inserting the lenses into the lens case basket holders and thoroughly rinsing them with Clear Care solution for at least 5 seconds before the 6-hour disinfection. In this study, the 5-second rinse step was able to slightly reduce the number of infectious adenovirus recovered from the lenses, but 10² infectious viral particles were still recovered even after 6-hour incubation with Clear Care solution. However, cleadew was able to reduce the number of infectious viral particles that adhered to both etafilcon A and lotrafilcon B to below the limit of quantification, even when the lenses were not rinsed with cleadew after the incubation period as recommended by the manufacturers. These results are in accordance with the quantitative suspension assays, in which PVP-I showed higher efficacy than 3% hydrogen peroxide against Ad5.

CONCLUSIONS

In this study, quantitative suspension tests suggest that products containing PVP-I (cleadew and cleadew GP) have a significantly higher disinfection efficacy than the other care products evaluated in this study. Surprisingly, 3% hydrogen peroxide was not efficient against Ad5, even when the manufacturer’s instructions were followed. It was also demonstrated that various contact lens materials, including conventional hydrogel, silicone hydrogel, and rigid lenses, were similarly prone to Ad5 contamination, which could be removed from the lens surfaces when rubbed or rinsed with only saline. Moreover, the efficacy of contact lens care products based on nonoxidative disinfection systems against Ad5 can be significantly improved by ensuring that a rub-and-rinse step is used prior to overnight disinfection, highlighting the importance of following the disinfection regimens of contact lens care products to remove infectious adenovirus from lens material surfaces.