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In Vitro Evaluation of the Virucidal Activity of Different Povidone–Iodine Formulations Against Murine and Human Coronaviruses

Infectious Diseases and Therapy volume 10, pages 2777–2790 (2021)Cite this article

Abstract

Introduction

Polyvinylpyrrolidone–iodine (PVP-I) demonstrates broad-spectrum anti-infective activity and is available in different formulations for oral rinse and topical use in medical and personal care settings. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has underscored the need to supplement available preventive strategies.

Methods

We assessed virucidal activity of PVP-I formulations, including 0.5% (w/v) solution, 5.0% (w/v) solution, 7.5% (w/v) scrub, and 10.0% (w/v) solution, versus placebos when challenged with coronaviruses in two in vitro studies. Murine coronavirus strain A59 (American Type Culture Collection [ATCC]® VR-764™), human coronavirus strain OC43 (ZeptoMetrix Corp. #0810024CF), human coronavirus strain NL63 (ZeptoMetrix Corp. #0810228CF), and human coronavirus strain 229E (ATCC® VR-740™) were used as surrogates for SARS-CoV-2. Both studies used the American Society for Testing and Materials in vitro time-kill method.

Results

All active PVP-I formulations in study 1 demonstrated virucidal activity at 15 s, with mean log10 reduction of greater than 4.56 or greater than 99.99% inactivation; a cytotoxic effect against the National Collection of Type Cultures clone 1469 host cells was observed with 5.0% (w/v) solution, 7.5% (w/v) scrub, and 10.0% (w/v) solution. Active PVP-I formulations in study 2 demonstrated effective virucidal activity against coronaviruses in less than 15 s; log10 reduction in viral titer for each coronavirus strain was consistently higher for 10.0% (w/v) solution and 0.5% (w/v) solution versus 7.5% (w/v) scrub.

Conclusion

Both studies demonstrated in vitro virucidal activity of PVP-I formulations when challenged with SARS-CoV-2 surrogate coronaviruses. Although promising, further investigations are needed to evaluate SARS-CoV-2 inactivation.

FormalPara Key Points
Why carry out the study?
The coronavirus disease of 2019 (COVID-19) pandemic underscores the need to supplement strategies to prevent virus transmission in home-based, community, and healthcare settings.
We assessed the virus inactivation effect of different povidone–iodine formulations, such as oral rinses, scrubs, and topical solutions, against surrogate viruses for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in two in vitro preclinical studies.
What was learned from the study?
Povidone–iodine demonstrated anti-infective activity, with all active formulations exhibiting effective in vitro virucidal activity.
These results provide further understanding of the virucidal activity of povidone–iodine and serve as a knowledge base for future studies, including those evaluating SARS-CoV-2 inactivation.

Introduction

Polyvinylpyrrolidone–iodine (povidone–iodine or PVP-I) is an antiseptic agent with broad-spectrum anti-infective activity against a variety of pathogenic microorganisms, such as gram-negative and gram-positive bacteria, bacterial spores, fungi, protozoa, and viruses [1,2,3,4]. Virucidal activity against enveloped and non-enveloped viruses has been demonstrated [2, 5,6,7], with a short contact time relative to other commercially available antiseptic agents [7]. The efficacy and tolerability profile of PVP-I compared with that of other agents, such as chlorhexidine gluconate, polyhexanide, and octenidine, has been well established [1,2,3, 8]. Regardless of clinical use spanning decades, there has been no or low documented resistance or cross-resistance [1,2,3,4]. Furthermore, the United States (US) Food and Drug Administration confirmed this position in its final rule on non-prescription antiseptic preparations intended for professional use in healthcare settings by not requiring resistance testing for certain active ingredients, including PVP-I [9]. This salient feature of PVP-I adds utility in the era of antimicrobial resistance. Various PVP-I formulations have been developed, including oral rinses, gargles, topical solutions, and surgical scrubs, and are available for topical or oral use in healthcare, dental, and household settings [2,3,4, 10].

Emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 and its spread globally over the following year have proven to be an unprecedented public health crisis [11]. At the peak, global daily cases were almost 1.5 million in December 2020, and global daily deaths were almost 18,000 in January 2021 [11]. The coronavirus disease of 2019 (COVID-19) pandemic has brought into focus the urgent need for effective physical interventions to mitigate virus transmission in home-based, community, and healthcare settings [12, 13], especially in the absence of definitive treatments and expanded access to available vaccines globally.

With a renewed focus on hand hygiene [12], evaluations of the virucidal activity of rinses, solutions, and scrubs have seen a significant uptake as the global community seeks to build an effective armamentarium against SARS-CoV-2. With respect to oral hygiene, PVP-I mouthwash was included in the list of non-drug experimental therapies against SARS-CoV-2 early in the pandemic [14]. Several in vitro studies have evaluated PVP-I formulations against SARS-CoV-2 and other viruses, including the Ebola virus and Middle East respiratory syndrome coronavirus (MERS-CoV) [5, 6, 15,16,17,18,19,20]. PVP-I formulations have been evaluated for use in high-risk clinical procedures involving the oropharynx and nasopharynx or in surgical practice [21,22,23] as a means to supplement available strategies, such as personal protective equipment [24], and prevent or reduce virus transmission during the ongoing pandemic.

Herein, we report findings from two in vitro studies that assessed the virucidal activity of different PVP-I formulations compared with matching placebos against the alphacoronavirus and betacoronavirus genera, which are surrogates for SARS-CoV-2. Study 1 assessed the efficacy of different PVP-I formulations against murine betacoronavirus strain A59 using the American Society for Testing and Materials (ASTM) in vitro time-kill method. Study 2 evaluated the virucidal properties of different PVP-I formulations challenged with human betacoronavirus strain OC43, human alphacoronavirus strain NL63, and human alphacoronavirus strain 229E using a virucidal suspension test (i.e., ASTM in vitro time-kill method).

Methods

Study Design and Rationale

Coronaviruses have originated from many different animal species, including bats, cattle, camels, civets, and rodents [25]. They are known to cause mild to serious respiratory, gastrointestinal, and neurological diseases. Efficient viral transmission appears to depend upon the viral proteins and unique cell surface receptor(s) on the invaded cell types. The present preclinical in vitro study was undertaken to better understand the broad-spectrum antiviral activity of PVP-I and to determine, if any, differential effectiveness for various PVP-I formulations against various coronavirus strains. In this study, the efficacy of different PVP-I formulations was compared with matching placebos against human and murine coronavirus surrogates for SARS-CoV-2.

Test Formulations

Different test formulations, including active PVP-I (Betadine®, Avrio Health L.P., Stamford, USA) and placebo, were assessed: 0.5% (w/v) solution, placebo 0.5% (w/v) solution, 5.0% (w/v) solution, 7.5% (w/v) scrub, placebo 7.5% (w/v) scrub, and 10.0% (w/v) solution in study 1 and 0.5% (w/v) solution, placebo 0.5% (w/v) solution, 7.5% (w/v) scrub, placebo 7.5% (w/v) scrub, and 10.0% (w/v) solution in study 2.

Virus Strains and Host Cells

In study 1, murine coronavirus strain A59 (American Type Culture Collection [ATCC]® VR-764™), a betacoronavirus from the same genus that includes SARS-CoV-1, SARS-CoV-2, and MERS-CoV, was used as a surrogate for SARS-CoV-2; the strain was sourced from Dr. Nerea Irigoyen (Department of Pathology, University of Cambridge, UK). Mouse hepatitis virus A59 was used as a surrogate test virus for SARS-CoV-2 in study 1 since it is a representative member of the genus betacoronavirus within the subfamily Coronavirinae-2, of which SARS-CoV-2 is a member [26,27,28]. The National Collection of Type Cultures (NCTC) clone 1469 cells (ATCC® CCL-9.1™; newborn mouse liver cells) were used as host cells. In study 2, test formulations were challenged with three human viral strains that served as surrogates for SARS-CoV-2 [29]: coronavirus strain OC43 (ZeptoMetrix Corp. #0810024CF), coronavirus strain NL63 (ZeptoMetrix Corp. #0810228CF), and coronavirus strain 229E (ATCC® VR-740™). MRC-5 cells (ATCC® CCL-171™; human lung fibroblast cells), Vero cells (ATCC® CCL-81™; green monkey epithelial kidney cells), and HCT-8 cells (ATCC® CCL-244™; human colon adenocarcinoma epithelial cells) were used as host cells.

Facilities

The assays were performed at separate facilities: study 1 at BluTest Laboratories Ltd., Glasgow, UK, and study 2 at BioScience Laboratories, Inc. (BSLI), Bozeman, Montana, USA. Standard equipment and supplies were used; calibration was in accordance with the standard operating procedure (SOP) of each facility. This article is based on in vitro studies and did not include research on human participants or animals performed by any of the authors.

Preparation of Test Formulations, Virus Suspensions, and Host Cells

For study 1, higher-concentration PVP-I formulations (i.e., 5.0% (w/v) solution, 7.5% (w/v) scrub, and 10.0% (w/v) solution) were diluted 1:10 in sterile distilled water for neutralization validation and the in vitro time-kill assay; the remaining test formulations (i.e., 0.5% (w/v) solution, placebo 0.5% (w/v) solution, and placebo 7.5% (w/v) scrub) were used undiluted. For study 2, all test formulations were used as received from the study sponsor; final test formulations had a concentration of 90%.

Virus strains were propagated and stored per standard procedure for production of high-titer virus stock (BSLI SOP L-2102); aliquots of stock virus suspension stored at −70 °C were thawed before use. ATCC-obtained host cells were maintained as monolayers in disposable cell culture labware in accordance with standard procedure (BSLI SOP L-2084). Before testing, these cultures were seeded onto multiwell, cell culture-treated plates. Vero and HCT-8 cell monolayers were 80–90% confluent and less than 48 h old before virus inoculation; MRC-5 cells were 80% confluent and approximately 51 h old. The growth medium (i.e., RPMI-1640 or 1X Eagle’s Minimum Essential Medium with 10% fetal bovine serum and 1% antibiotic and l-glutamine) was replaced with the maintenance medium (i.e., RPMI-1640 or 1X Eagle’s Minimum Essential Medium with 2% fetal bovine serum and 1% antibiotic and l-glutamine) to support virus propagation.

Assessment of Cytopathic/Cytotoxic Effect

Cytopathic/cytotoxic effect in host cells was monitored to determine the lowest concentration of PVP-I that was noncytopathic and noncytotoxic.

Neutralization Validation

Neutralization validation was performed to confirm the effectiveness of the procedure in neutralizing the active virucidal component with each formulation; this was performed for the median tissue culture infective dose (TCID50) input virus, positive control, negative control, and test formulations. Standard testing methods based on ASTM were employed. Neutralization was validated when virus recovery in the positive control matched that in the neutralized formulation.

Virucidal Suspension Test Using the Time-Kill Method

For study 1, triplicates were set up for each designated exposure time point. The first well contained 0.8 mL of the test formulation at a specific concentration and 0.1 mL of sterile water. The procedure was initiated when 0.1 mL of virus was added to this well and mixed. Samples were incubated for designated exposure times of 15 s, 30 s, 60 s, and 5 min. Upon the specified time indicated, a 0.1-mL volume from the well was sampled and applied gently to the surface of a prepared MicroSpin S-400 HR column and eluted per manufacturer’s instructions. The void volume eluent (0.1 mL) was applied to a well containing 0.9 mL of 5% bovine serum albumin in phosphate-buffered saline at 20 ± 1 °C and incubated for 5 min at 20  ± 1 °C. Following incubation, 0.1 mL of the mixture was diluted in 0.9 mL of cell culture media before being serially diluted up to a concentration of 10−6 and plated per the standard TCID50 reads/calculations; results were recorded. Subsequent analyses were performed using the well-established Spearman-Kärber method of quantification [30]. Positive controls were set up alongside the test formulation being processed; the positive-control well contained 0.8 mL of cell culture media, 0.1 mL of sterile water, and 0.1 mL of the virus. Positive controls were assessed at 0 s and 5 min; negative controls were assessed at 15 s and 5 min. The negative control runs were identical to the test formulation runs, except water was substituted for the virus suspension.

For study 2, a virucidal suspension test based on ASTM E1052-11 (Standard Test Method to Assess the Activity of Microbicides against Viruses in Suspension) was also performed. A 0.5-mL aliquot of test virus was added to 4.5 mL of undiluted test formulation to obtain a 90% (w/v) concentration of the test formulation. The challenge virus suspension was exposed to the test formulation for the designated exposure times (i.e., 0 [< 15 s], 15 s, 30 s, 60 s, and 5 min). Immediately after exposure, the test virus suspension was neutralized in Dey–Engley neutralizing broth, mixed thoroughly, and serially diluted in the maintenance medium. Each dilution was plated in four replicates.

Analytical Methods

Viral titers in both studies were determined using the quantal test (Spearman-Kärber method) [30] and expressed as TCID50 using the following formula: log TCID50 = L − d(s − 0.5)/inoculum volume, where L is the negative log10 of the lowest dilution, d is the difference between the dilution steps, and s is the sum of proportions of the positive wells per test dilution. Reduction in test virus infectivity of each strain was calculated as the difference in log10 TCID50/mL between the virus control and the test virus; this was done at the designated exposure times (i.e., 15 s, 30 s, 60 s, and 5 min for study 1 and 0 [< 15 s], 15 s, 30 s, 60 s, and 5 min for study 2). The percentage reduction was also calculated and reported.

Results

Study 1

All test formulations were validated for neutralization across triplicates at 60 s exposure time (data not shown). Active PVP-I formulations demonstrated effective virucidal activity against the murine coronavirus strain A59 at 15 s under test conditions. The mean log10 reductions in viral titer after exposure to test conditions are presented in Table 1. Three of the PVP-I formulations (i.e., 0.5% (w/v) solution, 5.0% (w/v) solution, and 10.0% (w/v) solution) demonstrated high virucidal activity against the murine coronavirus strain A59 at 15 s, with a mean log10 reduction of greater than 4.56 or greater than 99.99% viral inactivation; this was lower for 7.5% (w/v) scrub, which had a mean log10 reduction of greater than 3.56. Placebo 0.5% (w/v) solution showed the lowest virucidal activity against the murine coronavirus strain A59 (mean log10 reduction of 0.17 at 15 s). Mean log10 reductions in viral titer for all test formulations are illustrated in Fig. 1.

Table 1 Virucidal activity of six test formulations, including PVP-I and placebo, against the murine coronavirus strain A59 following designated exposures of 15 s, 30 s, 60 s, and 5 min (study 1)
Full size table
Fig. 1
figure 1

Mean log10 reductions in viral titer as a function of exposure time for the six test formulations, including PVP-I and placebo (study 1). PVP-I polyvinylpyrrolidone–iodine, w/v weight per volume

Full size image

Cytotoxic effects against NCTC clone 1469 host cells were observed with some of the PVP-I formulations (i.e., 5.0% (w/v) solution, 7.5% (w/v) scrub, and 10.0% (w/v) solution). Accordingly, these formulations were tested at 1:10 dilution instead as this was the lowest concentration of PVP-I that was noncytotoxic against NCTC clone 1469 host cells. Furthermore, 7.5% (w/v) scrub showed a lower log10 reduction at 1:10 dilution.

The efficacy of positive and negative virus controls was also evaluated for each test formulation at exposure times of 0 s and 5 min, and 15 s and 5 min, respectively. The positive controls for all test formulations, including active and placebo, showed mean log10 reductions in infectivity of 6.00 at 0 s and 6.06 at 5 min. For negative controls across all test formulations, no differences were observed in mean log10 reductions in infectivity for exposure at 15 s versus 5 min; mean log10 reductions in infectivity of 1.50 were observed for 0.5% (w/v) solution, placebo 0.5% (w/v) solution, 5.0% (w/v) solution, and 10.0% (w/v) solution; 2.50 for 7.5% (w/v) scrub; and 3.50 for placebo 7.5% (w/v) scrub.

Study 2

All test formulations were validated for neutralization along with neutralizer toxicity controls (data not shown). Across test formulations, the three PVP-I formulations demonstrated effective virucidal activity against the coronavirus strains in less than 15 s, with mean log10 reduction in viral titers and some variability by PVP-I formulation and coronavirus strain; the infective dose and mean log10 reductions in viral titer after exposure to test conditions are presented in Table 2. The 0.5% (w/v) solution reduced the infectivity of all three coronavirus strains; the mean log10 reduction was the highest within 15 s of exposure for coronavirus strain NL63 (4.75 log10 or greater than 99.99% viral inactivation). The 10.0% (w/v) solution also reduced the infectivity of all three coronavirus strains; the mean log10 reduction within 15 s of exposure was the highest for coronavirus strain NL63 (at least 5.25 log10 or greater than 99.99% viral inactivation). Viral inactivation within 15 s across all coronavirus strains was the lowest for 7.5% (w/v) scrub compared with the other active PVP-I formulations. The mean log10 reductions in viral titer for each coronavirus strain are illustrated in Fig. 2; these were consistently higher for 0.5% (w/v) solution and 10.0% (w/v) solution compared with 7.5% (w/v) scrub and placebos.

Table 2 Virucidal activity of five test formulations, including PVP-I and placebo, against coronavirus strains OC43, NL63, and 229E following designated exposures of 0 (< 15 s), 15 s, 30 s, 60 s, and 5 min (study 2)
Full size table
Fig. 2
figure 2

Mean log10 reductions in viral titer as a function of exposure time for the five test formulations, including PVP-I and placebo (study 2). Coronavirus strains a OC43, b NL63, c 229E. PVP-I polyvinylpyrrolidone–iodine, w/v weight per volume

Full size image

Under the experimental conditions used, cytopathic or cytotoxic effect against host cells was observed for some test formulations: for coronavirus strain OC43, 0.5% (w/v) solution for exposure of 0 (< 15 s), placebo 0.5% (w/v) solution for all exposure times, 7.5% (w/v) scrub for all exposure times, placebo 7.5% (w/v) scrub for all exposure times, and 10.0% (w/v) solution for exposure of 0 (< 15 s).

Discussion

Though PVP-I has been shown to be a broad-spectrum antiseptic agent, activity against murine and human coronavirus strains has not been previously reported in the literature. Our research using surrogate coronaviruses expands on the body of virucidal effectiveness data of PVP-I. Testing against surrogates helps inform several different research needs related to the novel coronavirus, including strain-to-strain comparisons, replicating test methods, and improving statistical confidence. Agent–surrogate comparison testing is also useful to ascertain if SARS-CoV-2 has unnatural environmental or antimicrobial persistence compared with other coronaviruses [31].

Our findings on virucidal activity were consistent with the results of other studies, including in vitro studies and an in vivo simulation study, of PVP-I formulations tested against viruses [5, 6, 15, 18, 32], and an observed log10 reduction in the viral titer of at least 4, per European Standards of disinfectant efficacy [33]. PVP-I virucidal activity was observed within or at 15 s and persisted until 5 min, the last measured exposure time in both studies, and was indicative of rapid and prolonged viral inactivation. Virucidal activity varied by PVP-I formulation: in study 1, it was higher for 0.5% (w/v) solution, 5.0% (w/v) solution, and 10.0% (w/v) solution against murine coronavirus strain A59 at designated exposure times compared with 7.5% (w/v) scrub; in study 2, it was consistently higher for 0.5% (w/v) solution and 10.0% (w/v) solution against three coronavirus strains compared with 7.5% (w/v) scrub. It must be noted that 7.5% (w/v) scrub exhibited a lower log10 reduction (i.e., less than 4 log10 reduction) at 1:10 dilution in study 1 compared with the other PVP-I formulations. This PVP-I formulation is designed as a sudsing cleanser for use in a medical/surgical setting. The sudsing is achieved through the inclusion of nonionic surfactants (e.g., nonoxynol-9). Following the mechanical action of handwashing, the cleanser and any associated pathogens are subsequently washed off the skin. Washing off the cleanser and associated pathogens in the clinical setting may be an important consideration in interpreting these in vitro study findings. Furthermore, it was hypothesized that some of the coformulant(s) may have contributed to host cell cytotoxicity as the undiluted placebo 7.5% (w/v) scrub, which had the same formulation but without active PVP-I, also demonstrated similar cytotoxic effect against NCTC clone 1469 host cells, potentially leading to lower log10 reduction values. In addition, since at least one of the nonionic surfactants in the formulation is known to exhibit microbicidal effects (e.g., nonoxynol-9) [34, 35], it is not surprising that the placebo 7.5% (w/v) scrub exhibited some antiviral activity in these studies.

While recognized as safe and effective, there is a lack of clear consensus on US guidance related to benchmarks for virucidal efficacy testing (i.e., the magnitude of reduction in viral infectivity) [36]. Technical guidance has been established in Europe (i.e., European Standards or EN) to define requirements for test methods, including in vitro testing methods to ascertain the disinfectant efficacy [37] of agents such as PVP-I, and the magnitude of log10 reduction in viral titer (at least 4) [33, 38]. This benchmark is often reported as evidence of the efficacy of disinfectant activity and for comparison of results [5, 18], including for various antiseptic agents. In one study that included PVP-I, triclosan, chlorhexidine digluconate, octenidine dihydrochloride, and polyhexanide, the optimal efficacy of PVP-I was demonstrated over shorter and more immediate contact times of 1 min [39]. Another study assessed the bactericidal and virucidal efficacy of PVP-I [7.5% (w/v) scalp and skin cleanser], chlorhexidine gluconate [4.0% (w/v) hand cleanser], and the reference handwash using the European Standard EN1499 and demonstrated a significantly higher mean log10 reduction with PVP-I compared with chlorhexidine gluconate for different volumes and contact times [32].

Physical interventions to reduce viral transmission during the current COVID-19 pandemic in healthcare, community, and home-based settings include maintaining hand hygiene by frequent and proper handwashing [12] and the use of personal protective measures [14, 24]. In vitro studies of PVP-I using challenge testing have assessed virucidal efficacy, including against coronaviruses such as SARS-CoV-2 [15,16,17,18,19]. In one study, all four PVP-I formulations (10.0% (w/v) antiseptic solution, 7.5% (w/v) antiseptic skin cleanser, 1.0% (w/v) gargle and mouthwash, and 0.45% (w/v) throat spray) achieved at least 99.9% viral inactivation or at least a 4 log10 reduction in viral titer within 30 s of contact [15]. In a study evaluating the in vitro virucidal activity of PVP-I gargle and mouthwash, the undiluted (PVP-I 1% w/v) and 1:2 dilution (PVP-I 0.5% w/v) formulations demonstrated rapid virucidal activity (at least 4 log10 reduction in viral titer) at 15 s [18]. Although a gap exists in the interpretation of findings from in vitro studies and evidence from clinical studies is needed, effective anti-infective therapy has the potential to contribute significantly to ensuring safer healthcare and household environments, especially in the current pandemic.

Through our in vitro studies, we demonstrated the virucidal activity of a variety of PVP-I formulations, such as gargle/oral rinse, solution, and scrub, at different concentrations and with complementary uses in medical, dental, or surgical settings and personal care settings, when challenge tested against alphacoronavirus and betacoronavirus strains that served as surrogates for SARS-CoV-2. It is encouraging to see recent preclinical and clinical evidence demonstrating PVP-I-mediated in vitro inactivation of SARS-CoV-2 [40,41,42,43,44,45] as well as reduction in viral load using PVP-I oral formulations in randomized clinical trials on patients with COVID-19 [46, 47]. Our findings and studies by other groups, together, strengthen the evidence for implementing the use of various PVP-I formulations in medical and personal care settings against a family of coronavirus strains, including SARS-CoV-2, as a broad-spectrum virucidal agent that neutralizes human and murine coronaviruses, potentially informing behaviors and preventing transmission during future pandemics. In these unprecedented times, the ongoing research using PVP-I provides additional safeguards to enhance existing preventive strategies and increase our armamentarium to prevent and reduce SARS-CoV-2 transmission in medical and community settings. To prevent virus transmission among patients, healthcare workers, and caregivers during the COVID-19 pandemic, future clinical studies on PVP-I effectiveness should include evaluation of the following: virucidal activity against SARS-CoV-2; optimal delivery methods or formulations, including oral rinse, intranasal spray, or scrub; and optimal concentrations of different formulations for SARS-CoV-2 inactivation. Our study has some limitations. First, the in vitro nature of the study design prevents clinical interpretation of the findings. Second, there is a paucity of US standards for evaluating the in vitro virucidal efficacy of anti-infective agents against an established benchmark related to the magnitude of log10 reduction in viral titer and infectivity. Such a benchmark would provide a much-needed tool to evaluate the virucidal activity or efficacy of disinfectants.

Conclusion

Both studies demonstrated the in vitro virucidal activity of different PVP-I formulations against coronaviruses using the time-kill method. While these in vitro findings are promising, they should serve as a basis for clinically focused investigations to generate evidence on optimal concentrations and delivery methods and to evaluate the virucidal activity of PVP-I formulations against SARS-CoV-2.

References

  1. Barreto R, Barrois B, Lambert J, Malhotra-Kumar S, Santos-Fernandes V, Monstrey S. Addressing the challenges in antisepsis: focus on povidone iodine. Int J Antimicrob Agents. 2020;56:106064. https://doi.org/10.1016/j.ijantimicag.2020.106064.

    CAS  Article  PubMed  Google Scholar 

  2. Eggers M. Infectious disease management and control with povidone iodine. Infect Dis Ther. 2019;8:581–93. https://doi.org/10.1007/s40121-019-00260-x.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Lachapelle J-M, Castel O, Casado AF, et al. Antiseptics in the era of bacterial resistance: a focus on povidone iodine. Future Med. 2013;10:579–92.

    CAS  Google Scholar 

  4. Williamson DA, Carter GP, Howden BP. Current and emerging topical antibacterials and antiseptics: agents, action, and resistance patterns. Clin Microbiol Rev. 2017;30:827–60. https://doi.org/10.1128/CMR.00112-16.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Eggers M, Eickmann M, Kowalski K, Zorn J, Reimer K. Povidone-iodine hand wash and hand rub products demonstrated excellent in vitro virucidal efficacy against Ebola virus and modified vaccinia virus Ankara, the new European test virus for enveloped viruses. BMC Infect Dis. 2015;15:375. https://doi.org/10.1186/s12879-015-1111-9.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Eggers M, Eickmann M, Zorn J. Rapid and effective virucidal activity of povidone-iodine products against Middle East respiratory syndrome coronavirus (MERS-CoV) and modified Vaccinia virus Ankara (MVA). Infect Dis Ther. 2015;4:491–501. https://doi.org/10.1007/s40121-015-0091-9.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Kawana R, Kitamura T, Nakagomi O, et al. Inactivation of human viruses by povidone-iodine in comparison with other antiseptics. Dermatol. 1997;195(Suppl 2):29–35. https://doi.org/10.1159/000246027.

    Article  Google Scholar 

  8. Kampf G, Todt D, Pfaender S, Steinmann E. Persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents. J Hosp Infect. 2020;104:246–51. https://doi.org/10.1016/j.jhin.2020.01.022.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Food and Drug Administration, Health and Human Services. Safety and effectiveness of health care antiseptics; topical antimicrobial drug products for over-the-counter human use (Docket No. FDA-2015-N-0101; final rule). Fed Reg. 2017;82:60474–503.

    Google Scholar 

  10. Leaper DJ, Durani P. Topical antimicrobial therapy of chronic wounds healing by secondary intention using iodine products. Int Wound J. 2008;5:361–8. https://doi.org/10.1111/j.1742-481X.2007.00406.x.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Johns Hopkins University. Coronavirus Resource Center. 2021. https://coronavirus.jhu.edu/map.html. Accessed 21 Apr 2021.

  12. World Health Organization. SAVE LIVES: Clean Your Hands Campaign. WHO. 2020. https://www.who.int/campaigns/save-lives-clean-your-hands. Accessed 13 Jan 2021.

  13. Ran L, Chen X, Wang Y, Wu W, Zhang L, Tan X. Risk factors of healthcare workers with coronavirus disease 2019: a retrospective cohort study in a designated hospital of Wuhan in China. Clin Infect Dis. 2020;71:2218–21. https://doi.org/10.1093/cid/ciaa287.

    CAS  Article  PubMed  Google Scholar 

  14. World Health Organization. COVID 19 landscape of experimental treatments. WHO. 2020. https://www.who.int/publications/i/item/covid-19-landscape-of-experimental-treatments. Accessed 4 Jan 2021.

  15. Anderson DE, Sivalingam V, Kang AEZ, et al. Povidone-iodine demonstrates rapid in vitro virucidal activity against SARS-CoV-2, the virus causing COVID-19 disease. Infect Dis Ther. 2020;9:669–75. https://doi.org/10.1007/s40121-020-00316-3.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Bidra AS, Pelletier JS, Westover JB, Frank S, Brown SM, Tessema B. Rapid in-vitro inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using povidone–iodine oral antiseptic rinse. J Prosthodont. 2020;29:529–33. https://doi.org/10.1111/jopr.13209.

    Article  PubMed  Google Scholar 

  17. Frank S, Brown SM, Capriotti JA, Westover JB, Pelletier JS, Tessema B. In vitro efficacy of a povidone–iodine nasal antiseptic for rapid inactivation of SARS-CoV-2. JAMA Otolaryngol Head Neck Surg. 2020;146:1054–8. https://doi.org/10.1001/jamaoto.2020.3053.

    Article  PubMed  Google Scholar 

  18. Hassandarvish P, Tiong V, Mohamed NA, et al. In vitro virucidal activity of povidone iodine gargle and mouthwash against SARS-CoV-2: implications for dental practice. Br Dent J. 2020;228:900. https://doi.org/10.1038/s41415-020-2402-0.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Pelletier JS, Tessema B, Frank S, Westover JB, Brown SM, Capriotti JA. Efficacy of povidone–iodine nasal and oral antiseptic preparations against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). Ear Nose Throat J. 2021;100(2_suppl):192S–196S. https://doi.org/10.1177/0145561320957237.

    Article  PubMed  Google Scholar 

  20. Siddharta A, Pfaender S, Vielle NJ, et al. Virucidal activity of World Health Organization-recommended formulations against enveloped viruses, including Zika, Ebola, and emerging coronaviruses. J Infect Dis. 2017;215:902–6. https://doi.org/10.1093/infdis/jix046.

    CAS  Article  PubMed  Google Scholar 

  21. Khan MM, Parab SR, Paranjape M. Repurposing 0.5% povidone iodine solution in otorhinolaryngology practice in Covid 19 pandemic. Am J Otolaryngol. 2020;41:102618. https://doi.org/10.1016/j.amjoto.2020.102618.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Mohamed NA, Baharom N, Wan Sulaiman WS, et al. Early viral clearance among COVID-19 patients when gargling with povidone–iodine and essential oils: a clinical trial. Int Med J. 2020;27:651–4. https://doi.org/10.1101/2020.09.07.20180448.

    CAS  Article  Google Scholar 

  23. Yan CH, Bleier BS. Prophylactic and therapeutic topical povidone-iodine in coronavirus disease 2019 (COVID-19): what is the evidence? Int Forum Allergy Rhinol. 2020;10:1271–3. https://doi.org/10.1002/alr.22735.

    Article  PubMed  Google Scholar 

  24. Mady LJ, Kubik MW, Baddour K, Snyderman CH, Rowan NR. Consideration of povidone-iodine as a public health intervention for COVID-19: utilization as “Personal Protective Equipment” for frontline providers exposed in high-risk head and neck and skull base oncology care. Oral Oncol. 2020;105: 104724. https://doi.org/10.1016/j.oraloncology.2020.104724.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Corman VM, Muth D, Niemeyer D, Drosten C. Hosts and sources of endemic human coronaviruses. Adv Virus Res. 2018;100:163–88. https://doi.org/10.1016/bs.aivir.2018.01.001.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Cui W, Cui S, Chen C, et al. The crystal structure of main protease from mouse hepatitis virus A59 in complex with an inhibitor. Biochem Biophys Res Commun. 2019;511:794–9. https://doi.org/10.1016/j.bbrc.2019.02.105.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Lissenberg A, Vrolijk MM, van Vliet ALW, et al. Luxury at a cost? Recombinant mouse hepatitis viruses expressing the accessory hemagglutinin esterase protein display reduced fitness in vitro. J Virol. 2005;79:15054–63. https://doi.org/10.1128/JVI.79.24.15054-15063.2005.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Yang Z, Du J, Chen G, et al. Coronavirus MHV-A59 infects the lung and causes severe pneumonia in C57BL/6 mice. Virol Sin. 2014;29:393–402. https://doi.org/10.1007/s12250-014-3530-y.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Chakraborty A, Diwan A. NL63: a better surrogate virus for studying SARS-CoV-2. Integr Mol Med. 2020;7:1–9. https://doi.org/10.15761/IMM.1000408.

    CAS  Article  Google Scholar 

  30. Ramakrishnan MA. Determination of 50% endpoint titer using a simple formula. World J Virol. 2016;5:85–6. https://doi.org/10.5501/wjv.v5.i2.85.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Guidance on SARS-CoV-2 surrogate selection (ASTM standard under consideration). ASTM. 2020. https://www.astm.org/COMMIT/GuidanceCOVID19SurrogateSel_April242020press.pdf. Accessed 10 Sep 2021.

  32. Eggers M, Koburger-Janssen T, Ward LS, Newby C, Müller S. Bactericidal and virucidal activity of povidone–iodine and chlorhexidine gluconate cleansers in an in vivo hand hygiene clinical simulation study. Infect Dis Ther. 2018;7:235–47. https://doi.org/10.1007/s40121-018-0202-5.

    Article  PubMed  PubMed Central  Google Scholar 

  33. European Committee for Standardization. EN 14476:2013+A2:2019 Chemical disinfectants and antiseptics—quantitative suspension test for the evaluation of virucidal activity in the medical area—test method and requirements (Phase 2/Step 1). 2019. https://standards.iteh.ai/catalog/standards/cen/10065fd9-2d40-4f02-bccd-48206b72f108/en-14476-2013a2-2019. Accessed 10 Sept 2021.

  34. Hooton TM, Fennell CL, Clark AM, Stamm WE. Nonoxynol-9: differential antibacterial activity and enhancement of bacterial adherence to vaginal epithelial cells. J Infect Dis. 1991;164:1216–9. https://doi.org/10.1093/infdis/164.6.1216.

    CAS  Article  PubMed  Google Scholar 

  35. Klebanoff SJ. Effects of the spermicidal agent nonoxynol-9 on vaginal microbial flora. J Infect Dis. 1992;165:19–25. https://doi.org/10.1093/infdis/165.1.19.

    CAS  Article  PubMed  Google Scholar 

  36. US Food and Drug Administration. FDA letter regarding deferral of povidone–iodine. 2017. https://www.regulations.gov/document?D=FDA-2015-N-0101-1326. Accessed 7 Jan 2021.

  37. Suchomel M, Kundi M, Pittet D, Rotter ML. Modified World Health Organization hand rub formulations comply with European efficacy requirements for preoperative surgical hand preparations. Infect Control Hosp Epidemiol. 2013;34:245–50. https://doi.org/10.1086/669528.

    Article  PubMed  Google Scholar 

  38. Suchomel M, Steinmann J, Kampf G. Efficacies of the original and modified World Health Organization-recommended hand-rub formulations. J Hosp Infect. 2020;106:264–70. https://doi.org/10.1016/j.jhin.2020.08.006.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Koburger T, Hübner N-O, Braun M, Siebert J, Kramer A. Standardized comparison of antiseptic efficacy of triclosan, PVP-iodine, octenidine dihydrochloride, polyhexanide and chlorhexidine digluconate. J Antimicrob Chemother. 2010;65:1712–9. https://doi.org/10.1093/jac/dkq212.

    CAS  Article  PubMed  Google Scholar 

  40. Davies K, Buczkowski H, Welch SR, et al. Effective in vitro inactivation of SARS-CoV-2 by commercially available mouthwashes. J Gen Virol. 2021;102: https://doi.org/10.1099/jgv.0.001578.

    CAS  Article  PubMed Central  Google Scholar 

  41. Xu C, Wang A, Hoskin ER, et al. Differential effects of antiseptic mouth rinses on SARS-CoV-2 infectivity in vitro. Pathogens. 2021;10:272. https://doi.org/10.3390/pathogens10030272.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Hassandarvish P, Tiong V, Mohamed NA, et al. In vitro virucidal activity of povidone iodine gargle and mouthwash against SARS-CoV-2: implications for dental practice. Br Dent J. 2020. https://doi.org/10.1038/s41415-020-2402-0.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Anderson DE, Sivalingam V, Kang AEZ, et al. Povidone-iodine demonstrates rapid in vitro virucidal activity against SARS-CoV-2, the virus causing COVID-19 disease. Infect Dis Ther. 2020;9:669–75. https://doi.org/10.1007/s40121-020-00316-3.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Bidra AS, Pelletier JS, Westover JB, Frank S, Brown SM, Tessema B. Rapid in-vitro inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using povidone-iodine oral antiseptic rinse. J Prosthodont. 2020;29:529–33. https://doi.org/10.1111/jopr.13209.

    Article  PubMed  Google Scholar 

  45. Tadakamadla J, Boccalari E, Rathore V, Dolci C, Tartaglia GM, Tadakamadla SK. In vitro studies evaluating the efficacy of mouth rinses on Sars-Cov-2: a systematic review. J Infect Public Health. 2021;14:1179–85. https://doi.org/10.1016/j.jiph.2021.07.020.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Guenezan J, Garcia M, Strasters D, et al. Povidone iodine mouthwash, gargle, and nasal spray to reduce nasopharyngeal viral load in patients with COVID-19: a randomized clinical trial. JAMA Otolaryngol Head Neck Surg. 2021;147:400–1. https://doi.org/10.1001/jamaoto.2020.5490.

    Article  PubMed  Google Scholar 

  47. Seneviratne CJ, Balan P, Ko KKK, et al. Efficacy of commercial mouth-rinses on SARS-CoV-2 viral load in saliva: randomized control trial in Singapore. Infection. 2021;49:305–11. https://doi.org/10.1007/s15010-020-01563-9.

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

The authors thank the participating staff at the two testing facilities, i.e., BluTest Laboratories Ltd., Glasgow, UK, and BioScience Laboratories, Inc., Bozeman, Montana, USA, for their contributions to the two in vitro studies.

Funding

The research (i.e., study 1 and study 2) was funded by Imbrium Therapeutics L.P., a subsidiary of Purdue Pharma L.P. Manuscript development, including medical writing support, and Rapid Service Fee were funded by Avrio Health L.P., a subsidiary of Purdue Pharma L.P., Stamford, Connecticut, USA. The sponsor was involved in study design; the collection, analysis, and interpretation of data; the writing of the article; and the decision to submit the article for publication.

Medical Writing and Editorial Assistance

Medical writing assistance in the preparation of this article was provided by Melissa Furtado, MPH, and Juliane Moloney, PhD, of Cactus Life Sciences (part of Cactus Communications). Editorial assistance was provided by Cactus Life Sciences (part of Cactus Communications). Support for medical writing and editorial assistance was provided by Avrio Health L.P., a subsidiary of Purdue Pharma L.P.

Authorship

All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published.

Author Contributions

Manjunath Shet contributed to investigation, writing—original draft, writing—review and editing, and project administration. Rosa Hong contributed to conceptualization, writing—original draft, and writing—review and editing. David Igo contributed to resources, writing—original draft and writing—review and editing. Marc Cataldo contributed to writing—original draft and writing—review and editing. Sailaja Bhaskar contributed to conceptualization, writing—original draft, writing—review and editing, and supervision. All authors approved the final draft of the submitted manuscript.

List of Investigators

Dr. Christopher Woodall (Study Director for study 1) and Dr. Volha Teagle (Study Director for study 2).

Disclosures

All authors, i.e., Manjunath Shet, Rosa Hong, David Igo, Marc Cataldo, and Sailaja Bhaskar, are employees of either the company that markets and distributes Betadine® (povidone–iodine) antiseptic products (Avrio Health L.P.), the parent company (Purdue Pharma L.P.), or another subsidiary company of Purdue Pharma (Imbrium Therapeutics L.P.).

Compliance with Ethics Guidelines

This article is based on in vitro studies and did not include research on human participants or animals performed by any of the authors.

Data Availability

The data that support the findings of the two in vitro studies are included in this published article. Further information about datasets generated and/or analyzed during the two in vitro studies may be available for non-commercial purposes from the corresponding author on reasonable request.

Author information

Authors and Affiliations

  1. Imbrium Therapeutics, 201 Tresser Blvd, Stamford, CT, 06901, USA

    Manjunath Shet, David Igo & Sailaja Bhaskar

  2. Avrio Health L.P., 201 Tresser Blvd, Stamford, CT, 06901, USA

    Rosa Hong

  3. Purdue Pharma L.P., 201 Tresser Blvd, Stamford, CT, 06901, USA

    Marc Cataldo

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  1. Manjunath Shet
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  2. Rosa Hong
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  3. David Igo
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  4. Marc Cataldo
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  5. Sailaja Bhaskar
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Corresponding author

Correspondence to Marc Cataldo.

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Shet, M., Hong, R., Igo, D. et al. In Vitro Evaluation of the Virucidal Activity of Different Povidone–Iodine Formulations Against Murine and Human Coronaviruses. Infect Dis Ther 10, 2777–2790 (2021). https://doi.org/10.1007/s40121-021-00536-1

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Keywords

  • Anti-infective agents
  • Coronavirus
  • Povidone–iodine
  • SARS-CoV-2
  • Virus inactivation