Introduction
During the past 2 years, a viral pandemic with a novel human coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) lead to a global public health crisis. This circumstance caused a high impact on daily life, including dentistry and oral hygiene issues [
1]. The virus SARS-CoV-2 is mainly located in the nasopharyngeal tract as a main source for transmission, while the oral cavity and saliva also contains a certain amount of viral load, which is, however, of little value for airborne transmission of the virus [
2]. Accordingly, oral hygiene issues were repeatedly and comprehensively discussed in context of the current pandemic situation. On the one hand, usage of mouthwashes to reduce the viral load, and thus, the risk of transmission is an issue of high interest [
3]. On the other hand, oral hygiene aids were reported as potential habitat for SARS-CoV-2, increasing the risk of transmission. Thereby, oral hygiene aids were reported as potential way to spread the SARS-CoV-2 infection to cohabitating individuals [
4]. However, until now, there is no evidence on the transmission risk of viruses via oral hygiene aids.
Generally, the toothbrush has been examined as a potential source of microorganisms in different studies, including both bacterial and fungal species [
5‐
8]. While toothbrushes are commonly used for daily oral hygiene, their potential risk as a source of infection opens a new view on a potentially neglected health risk [
9,
10]. Toothbrushes of both, healthy and diseased individuals, become contaminated with oral bacteria, especially originating from dental plaque [
9]. Furthermore, other bacteria or fungi might also colonize the toothbrush, serving as a habitat for (self-) infection [
11]. Therefore, a variety of disinfection approaches to remove microorganisms from toothbrushes have been developed and evaluated, including microwave cooking, vinegar, alcohol, oral antiseptics, and UV light [
12‐
14]. Most of those approaches focused on the removal of bacteria from toothbrushes, showing mainly effective disinfection results [
12‐
14]. However, against the background of the current pandemic situation, the potential necessity of disinfection of toothbrushes to reduce viral load would be of interest, too. As first step to answer this question, it would be essential to investigate, whether viruses would be detectable on toothbrushes in an amount, which could potentially lead to a (self-) transmission. In this context, the effect of water rinsing and air-drying, as commonly performed after toothbrushing, would be needed to be considered.
Accordingly, this in vitro study had two aims: (I) it was examined whether two different viruses of importance and known to be transmitted via respiratory aerosols, i.e., coronavirus and influenza virus, would be detectable in a clinically relevant load on different parts of the manual toothbrush, depending on the time of air-drying. In this study, the feline coronavirus and the avian influenza A virus H1N1 were used. (II) It was also tested, if rinsing with water could reduce the viral load on the manual toothbrush. For this, a common high-quality standard protocol for testing viral load was applied. To differentiate the findings, different areas of the toothbrush were examined. It was hypothesized that viral load would be detectable at all parts of the toothbrush, while both air-drying and water rinsing lead to elimination of the titer of both viruses.
Discussion
The current worldwide pandemic situation shows the danger of the speed of spread of viral pathogens, which should not be underestimated. In the current event of a respiratory virus such as SARS-CoV-2, potential spread cycles must also be recognized on an everyday scale. In this context, dental hygiene plays a role, in addition to the already generally established measures such as personal hand hygiene. Aside of this way of infection, the transmission via contaminated surfaces has been thoroughly discussed [
15]. In order to assess the stability of important respiratory viruses on products of dental hygiene like toothbrushes and the associated (re)infection risk, contamination experiments with a coronavirus (FeCoV) and an influenza virus (AIV H1N1) were performed in this study.
For this purpose, controlled contamination experiments of different toothbrush areas (bristles, back or fixation) were performed to analyze virus tenacity. It was found that the titers of both viruses were rapidly and steadily reduced over the 24 h of the experiment. This reducing effect was particularly rapid on the contaminated toothbrush fixation. Furthermore, already within 12 h, an effective titer reduction of 2.5–5 log10 (FeCoV) and 2–4 log10 (H1N1) could be detected on all tested toothbrush parts. The residual titer was just above the detection limit at this point and changed little over the remainder of the experiment. Thus, both viruses show a low stability, which is further reduced by rinsing of the contaminated toothbrush parts, where no active virus could be recovered.
Meanwhile, different studies report a certain stability of the infectivity of coronaviruses, especially SARS-CoV-2 on surfaces [
15]. Interestingly, a previous study showed coronaviruses to remain infective on plastic surfaces (which is also the material of most toothbrushes) for hours, showing viable virus for up to 72 h after application [
16]. However, this previous examination showed very low titers after such a long observation period, while the titers after 12 and 24 h were similarly low as in the current study [
16]. Therefore, although a certain stability of the virus was detectable, the infectivity of the contaminated surface is very low, making a transmission unlikely. In case of the current study, the air-drying at room temperature seems to lead to a remarkable and fast reduction of the titer (i.e., below the limit of detection). Another study showed that the stability of coronavirus is remarkable reduced at 20 °C [
17]. Thus, the room temperature and laminar flow appear to lead to fast evaporation of the droplets and thus decreased viral load. Causal for this, the alteration of the envelope of the coronavirus because of continuous air-drying would be a plausible explanation for the fast loss in its infectivity. With regard to the clinical reality, a toothbrush is regularly rinsed with water after use. This was an experiment in the current study, resulting in a complete loss of virus load after rinsing and subsequently 12 h air-drying. As a result, the toothbrush is no habitat with a high risk of self-infection. It seems more plausible that patients, using the same toothbrush transmit the virus to each other, because of a generally reduced health behavior, for which using the same toothbrush could be an indicator. In this context, patients regularly use toothpaste for toothbrushing; toothpaste has an antimicrobial effect; although this was mainly shown for bacteria, the influence of the contamination of the toothbrush remains unclear [
18,
19].
FeCoV was selected as an alternative test virus for SARS-CoV-2 in this study. Both viruses belong to the family of
Coronaviridae; those viruses have an enveloped spherical structure with a diameter between 60 and 160 nm, while Influenza virus belongs to the family
Orthomyxoviridae, having an enveloped pleomorphic structure with a diameter ranging from 100 to 120 nm [
20]. This so-called surrogate virus method has long been used for efficacy testing of chemical disinfectants according to the guidelines of the German Veterinary Society (DVG). For this purpose, FeCoV and other viruses are commonly used as surrogates for related viruses and the results are directly transferred to the original viruses.
In addition to a coronavirus, the current experiment was also performed with another respiratory virus with high clinical relevance: Avian Influenza A virus H1N1. AIV H1N1, which caused a pandemic in 2009 [
21], is a type A influenza virus, an enveloped RNA virus, having a pleomorphic appearance with an average diameter of 120 nm [
22]. Influenza viruses are of high clinical interest, as they have caused hundreds of thousands of deaths worldwide each year [
23]. Moreover, in the current pandemic, influenza and SARS-CoV-2 are co-existing viruses, needing a joint preventive approach [
24]. Against this background, the same research question was applied for H1N1 in the current investigation, showing comparable results as for FeCoV. In turn, a study by Oxford et al. found that H1N1 is still infectious after 24 h on a plastic surface [
25]. During this study, H1N1 showed slightly lower titer reduction in the air-drying experiment than FeCoV; however, the titer after 12 and 24 h was low. Furthermore, the results after water rinse, again, corresponding to the clinical situation, were equal. Therefore, the toothbrush was also identified to be no important source of H1N1 and thus probably influenza virus (self-) transmission.
In general, a higher viral load of both viruses on bristles and bristle fixation would have been expected, based on the higher surface size. Nevertheless, the back of toothbrush head was found to show the comparably highest load. This might be explained by its smooth, non-porous surface, allowing a certain stability of the droplets and a slower evaporation, resulting in less collapse of the envelope of the virus. This, however, remains speculative and cannot be finally confirmed by the current data. The toothpaste, brushing technique, the toothbrush type (design and number of bristles, powered toothbrush, etc.), and interactions with salivatory components may limit the generalizability of the findings.
The study only included traditional toothbrush. More in-depth research on dental hygiene routines that deviate from the lab standard applied in this study is needed, for example, with electric toothbrushes or toothbrushes made of different materials (e.g., bamboo or wood) or more frequent brushing cycles.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.