Clinical Studies

More on the contaminated toothbrush: the viral story

  • Richard T. Glass, DDS, PhD*/ Harold G. Jense, PhD**


In early 1980s, while investigating various contributing factors in oral inflammatory diseases, one of us (RTG) found that patients with diseases such as recurrent oral ulcers (aphthous ulcers), burning mouth syndrome, migratory glossitis, and even advanced periodontal disease had a substantial decrease in both initial and recurrent symptoms simple by changing their toothbrushes. At first, patients were asked to change their toothrushes monthly. Later, patients were given four toothbrushes at each monthly appointment and were told to discard each toothbrush each brush after one week's use. Further improvement in these patients was noted. The clinical observations were substantiated by a systematic study of the role of toothbrushes as being a source of possible infection/reinfection. Similar studies have now been conducted in animals and these studies are in the final analysis. Concurrent with our discoveries, interest in both the scientific and the lay community has been focused on transmission of disease. While concern has been expressed in the wide range of media about the human immunodeficiency viruses (HIV-1, HIV-2), concern has also centered on then hepatits viruses and the herpes viruses. In single recent issue of the Journal of the American Medical Assocation , four separate articles dealt with herpes simplex viral infections (HSV-1, HSV-2)2-5. In one report2, there appeared to be an increased risk of HIV infections in those who were antibody-postive for HSV-2. Similarly, HSV-2 was found to be responsible for approximately one third of the cases of oral infections, presumable because of an increase in orogential sexual activity. 6. The same article also pointed out the presence of cytomegalovirus (CMV); Epstein-Barr virus (EBV); herps simplex virus type-1 (HSV-1); hepatitis A, B, non-A, and non-B (HAV; HBV; non-A, non-B), and HIV in the saliva. It is also clear that there is a growing population of medically compromised patients. Improved dental care has been successful in decreasing oral complications of cancer patients. 8. Reports continue to indicate that orally based bacteremia occur and that such bacteremia may be important in the death of medically compromised patients9. Finally, while there has been a recent rash of new toothbrushes, some with rather unusual designs and each remarkable claims, the basic toothbrush has changed little in recent years. Most of the changes have been in materials rather than design. No mention is made about efforts to lessen contamination of the toothbrush itself. All of the aformentioned seemingly unrelated findings come together in two questions: (1) Can the toothbrush harbor not only bacteria but also viruses? (2) Can these viruses be transmitted by such a source? In order to begin to answer these questions, studies were conducted to determine whether toothbrushes can be infected with viruses and whether these organisms can be passed by such an instrument.

Method and materials

In order to test the stability of herpes simplex virus type-1 (HSV-1) on toothbrushes,12 toothbrushes were immersed in 3 ml of 105.5 tissue culture infective dose (TCID) 50/0.1 ml in Hank's basic salt solution (HBSS). Two toothbrushes each were subjected to the following conditions: 1. Immersed for ten minutes, removed, rinsed, and :
A. Vortexed immediately
B. Placed in HBSS for 24 hours at room temperature (RT) and vortexed.

C. Placed in petri dishes at RT for 24 hours and vortexed
D. Placed in petri dishes at RT for 24 hours, placed
in HBSS at HBSS for 24 hours and vortexed

2. Immersed for 24 hours, removed, rinsed, and:
A. Vortexed immediately
B. Placed in HBSS for 24 hours and vortexed
For vertexing, the toothbrushes were placed in conical centifuge tubes containing 3 ml of HBSS and vortexed for 45 seconds.

The virus was quantitated on Vero cells (monkey kidney cells) grown in 96-well microtiter plates. The vortexed HBSS was diluted ten-fold in Eagle's minimal essential medium, supplemented with 2% newborn calf serum and gentamican (50 ug/ml). Quadruple, 0.1-ml samples were inoculated for each dilution. The microtiter plates were observed for up to five days for cytopathic effects of HSV-1 infection, and the results were recorded. (Fig.1) In order to test the role of toothbrush design and the effects of such factors as temperature, humidity, and rinsing, toothbrushes from varying manufacturers and varying design s were immersed in 3ml of 105TCID50/0.1ml in HBSS for ten minutes and placed in petri dishes at either RT or 28 C (in a nonhumdified incubator). Brushes were also reimmersed in HBSS (after initial ten-minutes exposure) and retained in this medium for 24, 48, 72, and 168 hours. After the specified time, all brushes were vortexed for 45 seconds and processed as in the first experiment. Both negative and controls were performed. Also vital staining of the virus on the toothbrushes was performed with Pap smear methods, and the toothbrushes were examined with the use of a dissecting microscope.


The results of the first experiments are summarized in Table 1. The data from the toothbrushes that were vortexed immediately demonstrated that 45% to 64% of the original inoculum could be retrieved from these toothbrushes. A substantial amount of the virus (18% to 64%) could be retrieved after the brushes were placed in HBSS for 24 hours, suggesting little occurrence of virus loss or death when the brushes were maintained in the moist environment. Toohthbrushes kept in a moist environment HBSS for 48 hours yielded 9% of the original inoculum. If the brushes were allowed to dry for 24 hours at room temperature, 27% to 45% of the original inoculum could be retrieved. When rehydrated for 24 hours in HBSS, these brushes yielded 18% to 27% of the original inoculum. The results of this study suggest that the difference in the retrieved inoculum might be a factor of either bristle type, bristle proximity, or overall brush design, coupled with factors such as humidity, and temperature and duration of storage. The results of the second experiment reveal that simple brush rinsing may decrease the amount of retrievable virus (Table 2). Similarly, different toothbrush designs appear to retain/retrieve more or less of the virus, depending on the number of tufts and the number of bristles per tuft. The fewer tufts and the fewer britsles per tuft probably have capillary action, thus absorbing and/or retaining less of the virus. The vital staining of the brushes demonstrated virus retention near the surface and along the shafts of the bristles. Microscopic examination of the bristles revealed the organism to be adherent, both to the smooth surfaces of the bristles and the to defects (irregularties) of the bristles. Natural toothbrushes were found to have many sharp edges on the bristle surface and a concentration of the virus in the central core of the bristle (Fig. 2). Finally, as is demonstrated in Table 3, in a moist environment, almost half of the virus could be retrieved after seven days. Once again, the length of survival/retrieval of the virus appears dependent on toothbrush design, especially proximity and the number of bristle tuft and bristles per tuft.


     In order to determine whether viruses could retained on toothbrushes and, further, whether they could be transmitted by a device, toothbrush from varying manufacturers were exposed to a known concentration of herps simplex virus-1. The virus was found to be retained on toothbrushes, the number of organisms surviving being dependant on such factors as humidity, temperature, and whether the toothbrush was rinsed after exposure to the virus.

    The virus was found to be transmissable and infectious by the measurment process (eg, the virus could infect and kill Vero cells). It is also clear from the results that toothbrush design plays a major role in the retention/retrieval of the virus. As demonstrated in both culture and vital staining, the organism was found both in the bristel defects and the smooth surfaces. We have had similar results in the previous experiments, using Candida albicans as our infecting organism.10

     Similarly, an increased number of tufts and an increased number of bristles per tuft increased the retention/retrieval of the virus. Historically, no mention in the literture was found regarding a biological basis for bristle tuft design. Microscopic examination of the toothbrushes from many manufacturers revealed frequent sharp or jagged edges of bristle ends. This finding was more common in natural-bristle brushes and brushes that did not claim bristle-end smoothing or beveling. Yet, even in those brushes which had bristle-end manipujlations, some bristle remained jagged, and these were the ones that tended to retain microorgansima.

     These same sharp bristle could theoretically lacerate the soft tissue of the gingiva or the mucosa, providing a portal of entry for the virus. With this information available, recurrent herps labiitis patients have been encouraged to change their toothbrushes during either the prodrome or the vesicular stage of the disease. They are again asked to change their toothbrushes either three days later or after the visicle breaks.

     Early reports from these patients indicate that either the lesion do not develop, or if they do develop, they do not spread. While this report deals with the herps simplex virus-1, it would also seems applicable to other virus. The HSV-1 is not the most resistant virus know to produce disease, but it is also not the most sensitive to death.6. In a pratical sense, it appears that the toothbrush may be responsible for either viral reinfection or the spread of viral infection to other indiviuals, similar to that found with other microorgansims.1.11. From these findings, is would seem reasonable to po]stulate that the spread of cold and flu viruses through a family can occur via toothbrush/toothpastecontamination.

     Finally, several recommendations seem advisable to lessen the possibilty of virual reinfection or spead of viral infections. First, the toothbrush should be changed on a regular basis. In previous studies, we suggested changing toothbrushes every month for healthy individuals. Considering the longevity of viruses, it may be more appropriate to change the toothbrush every two weeks. For medically compromised patients, toothbrush change weekly or every three days may be appropriate. Each member of the family should have his or her own toothpaste tube; these tubes should be small and changed with toothbrush frequency.

    Given the virus temperature-and-humidity-sensitivity, it is recommended that the toothbrush not be stored in the humid and contaminated environment of the bathroom. Rather, it should be stored vertically, with the bristle up, preferably in a less humid environment such as the bedroom. The brush should not be covered or enclosed because this may prevent the bristles from drying. While some chemical disinfectants will kill the HSV-1 on the toothbrush, these same chemicals do not kill all pathgens10. Lack of complete killing has also been found true of microwave sterilization , soaps, and boiling water. The latter methods also tend to distort and/or destroy the tootbrushses. Ultimately, toothbrushes should be designed in such a way microorganism retention is lessened.


It is apparent that HSV-1 can remain viable on a dried toothbrush for at least 48 hours and in a moist envronment for more seven days. The organism appears to be transmissible in that it can infect and produce cytotoxicity in Vero cells. This study provides more support for recommendations of regular toothbrush changing and immediate toothbrush changing in the presence of disease. Further, the storing of the toothbrush between uses should be in the dryest non-contaminated environment possible.


Studies have been conducted to determine the level of bacteria following oral hygiene procedures. A study by Sconyers et al. of toothbrushing in clinically healthy patients found no circulating bacteria in any of the 50 persons tested.1 In a study of similar design, Silvers et al. tested 36 subjects, 3 of whom exhibited detectable bacteremias.2 Both studies relied on 10 ml blood samples taken from the disinfected anticubital fossa during the last 30 seconds of brushing, and in both studies aerobic and anerobic cultures were obtained. Sconyers used an electric toothbrush for four minutes and Silver used a manual sulcular brush for two minutes. Only the Sconyers study, however, obtained a 10 ml prebrushing control blood sample. 

     These studies were conducted to determine the level of risk involved in normal oral hygiene procedures for high risk patients, such as rheumatic fever or congenital heart defects, or those with intracardiac and vascular prostheses. The relationship of bacteremia to toothbrushing in patients with periodontitis was also studied.3 Thirty periodontal patients were selected for the brushing study. As a baseline study, nine patients who were having extractions participated. Blood samples were drawn during the fourth minute of brushing and immediately after extraction procedures. Bacteria found in blood samples from the brushing group were compared with the bacteria cultures from the extraction group. Bacteremia was found in 5 of the 30 brushing patients and in all nine extraction cases.

     In the above mentioned studies, emphasis was placed on the disease or clinical condition in the patient's mouth as the factor affecting the bactermia. As early as 1920, Cobb cited the toothbrush as a cause of repeated infections of the mouth and reported on a significant case.4 Svanberg found that toothbrushes and toothpaste can be heavily infected with Streptococcus mutans for 24 hours after usage (the longest period tested was 24 hours). Svanberg also suggested that brushing with a contaminated brush introduces new microorganisms while simultaneously reducing existing normal flora.5

     Discussion of the modern toothbrush has suggested the problem of toothbrush construction as a factor of toothbrush contamination. The nylon multi-tuft toothbrush has been cited for its design of tufts set too closely to accommodate easy cleaning.6 With natural toothbrushes, the bristles can harbor inherent microorganisms. The natural toothbrush bristle has a central core or medulla running throughout the length of the bristle. When the brush is trimmed, the end of the bristle has an irregular shaped lumen. Fluids can be drawn into this core by capillary action, allowing for bacterial growth. the bristles also split longitudinally, further increasing the bacterial contamination.7

     The handles of modern toothbrushes are usually made of thermoplastic material, most commonly cellulose acetate, styrene acrylonitrile, or cellulose propionate. Nylon is almost universally used for filaments. The filaments are collected into bundles, bent in half with a metal anchor in the center, and driven into premolded holes in the toothbrush head at high speed. Nickel silver is used as anchor material because it is resistant to corrosion from toothpaste materials and from saliva residues. Despite the name "nickel silver," the alloy contains only nickel and copper.

     Several years ago, we observed that patients who had oral inflammatory disease tended to respond better to therapy if they had their old toothbrush replaced with a new one on a regular basis (e.g. replacement every two weeks). Further, when the inflammatory disease involved the tongue, necessitating tongue brushing, separate toothbrushes were used for the teeth and tongue. This observation prompted the following study to determine if toothbrushes harbor pathogenic microorganisms and if there is a correlation between contaminated brushes and the presence of disease.

Materials and Methods

The data for this study were collected from 30 synthetic toothbrushes. Ten new brushes from two manufacturers were cultures to determine whether microorganisms were present with a packaged brush. Ten brushes were cultured from clinically healthy patients and ten from patients with oral disease. A patient was considered "clinically healthy" if there were no caries, no mucosal abnormalities, and no gingival or periodontal inflammation. A patient was considered to have oral disease if he demonstrated large and/or numerous carious lesions, periodontal disease or mucosal disease (e.g., lichen planus, desquamative gingivitis, benign mucous membrane pemphigoid, burning mucosa, geographic tongue).

     The patients were asked to bring their toothbrushes to their dental examinations. Consent was obtained, a screening examination was performed, and significant data (toothbrush environment, oral health status, etc.) were collected.

     The toothbrush heads were transferred to sterile tubes (15 ml, orange-top centrifuge tubes with screw caps*) with an aseptic technique. Toothbrush handles were severed with end-cutting nippers which had been stored in Cidex (Formula 7).** Brushes were collected only during morning clinic hours to avoid drying of bristles. In oral-diseased patients, the specifically involved areas were also swabbed, and the specimens were immediately carried to the pathology lab for culture procedures.

     In order to again decrease the chances of contamination from brush transfer, the brushes and swabs remained in the sterile tubes, and brain-Heart Infusion Broth* was added. the borth was incubated at 37C until a turbid state existed. the following six plates were then inoculated and incubated at 37C:

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