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More on the contaminated toothbrush: the viral story
Introduction
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.
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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.
Results
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.
Discussion
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.
Conclusions
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.
Introduction
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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