Clinical Studies

Evaluation of the Use of the Purebrush Toothbrush Sanitizing Device in Immune-compromised Pediatric Patients
A Pilot Study

(Proposed Study Under Review)

Michael P. Link, MD, Principle Investigator*
Service Chief, Pediatric Hematology-Oncology
Professor of Pediatrics
Stanford University Medical Center
Tina Baggott, RN-CS, MN, PNP, CPON**
Pediatric Nurse Practitioner
Pediatric Hematology-Oncology
Stanford University Medical Center
R. Thomas Glass, D.D.S., Ph.D. ***
Professor of Pathology and Dental Medicine
Adjunct Professor of Microbiology
Chair, Department of Forensic Sciences
Director of the Forensic Sciences Graduate Program

300 Pasteur Dr.
Rm S304, MC:5208
Stanford, CA 94305
**Pediatric Hematology-Oncology
1000 Welch Road, Suite 300
Palo Alto, CA 94304-1812
***Oklahoma State University Center for Health Sciences
1111 West 17th Street
Tulsa, OK 74107-1898


In the early 1980’s, Glass, an oral and maxillofacial pathologist observed that culturing patients with recurring oral infections (ulcerations, burning mouths, and glossitis) and defining their offending microorganism(s) allowed for successful treatment with appropriate anti-microbial agents (Personal communication). When the treatment was removed, however, the infections recurred, suggesting that a disease transmission factor was missed in the treatment plan. Glass proposed that the missing factor might be the toothbrush.

The initial toothbrush study involved two populations of patients: 10 clinically normal individuals (positive controls) and 10 patients who had either mucosal or periodontal disease (study group). Each patient was asked to wrap the toothbrush after the last use and bring it to the clinic. As negative controls, two leading brands of toothbrushes were taken directly from the package. The toothbrushes were handled in an aseptic manner and cultured using an enriched microbial media (thioglycollate). The two predominating microorganisms were isolated and identified for each toothbrush. The results showed a wide array of microorganisms, many of which had been the same as those cultured in patients with mucosal and periodontal diseases. The difference between the control and the study group was that the concentration of microorganisms was higher in the study group. This study also showed that many of the microorganisms found on the toothbrush were considered as opportunistic or pathogenic for not only dental and mucosal diseases, but also for the respiratory and gastrointestinal tracts. What was completely unexpected was the finding that all five unused toothbrushes from one manufacturer had no microbial growth, while four out of five of the unused toothbrushes from a second manufacturer were contaminated in the package.

Once the concept that repeated use of toothbrushes allowed for contamination, the next study dealt with where the contamination actually occurred on the toothbrush itself. Toothbrushes were contaminated with Candida albicans in vitro by immersing the toothbrushes 106 microorganism/ml for 24 hours. The toothbrush bristle-ends were streaked across Sabouraud’s dextrose agar (specific for C. albicans). The toothbrush bristles were then sectioned mid-bristle length in an aseptic manner with a razor blade and were again streaked across the media. Finally, using sterile end-cutting nippers, the toothbrush heads were sectioned and touch to the media. The unexpected finding from this study was that the microorganisms were not just on the bristle end, but were also in the mid-portion of the toothbrush and in the head. Qualitatively, the concentration of microorganisms appeared to be as dense in all three areas. A caveat of this study (unpublished data) was that when toothbrushes were immersed in 106 microorganisms/ml for three minutes and allowed to stand in the open air similar to patient use, the toothbrushes yielded a concentration of 108 or 109 microorganisms/ml after 24 hours of incubation. Thus, the study indicated that that yeasts could not only contaminate the entire toothbrush, but could actually increase in numbers over a 24-hour period.

Finding that both bacteria and yeasts could adhere to toothbrushes, the next step was to look at viruses and the toothbrush. The first virus that was studied was the herpes simplex virus type-1 (HSV-1). HSV-1 is a DNA virus that is known to cause not only a generalized stomatitis in infants and children, but recurring lip lesions, conjunctivitis, encephalitis, herpetic whitlow, esophagitis, pneumonia, and disseminated infections in the adult. Twelve ethylene oxide sterilized toothbrushes were immersed in 3 ml of 105.5 tissue culture infective dose (TCID50) of HSV-1, with some toothbrushes being subsequently placed in a moist environment simulating the bathroom. This study showed that both the number of bristles/tuft and the number of tufts/toothbrush played a role in contamination by virus. The higher concentrations of virus were found in those toothbrushes with the highest number of bristles/tuft and the highest number of tufts/brush. Even more alarming was the finding that when toothbrushes were contaminated at a level of 105.5 TCID50 and subsequently stored in a moist environment simulating the bathroom for 168 hours (7 days), the toothbrushes retained 102.3 TCID50 viable viruses. The only reasonable conclusion from this study was that toothbrushes contaminated with HSV-1 could transmit the disease more than 7 days after initial contamination. This same study was repeated using a number of other pathogenic viruses with basically the same results.

Similarly, a small double blind-study of toothbrushes from HIV+ and HIV- patients was also conducted. Because HIV is an RNA virus, HIV proviral DNA was sought on ten toothbrushes. When the origin of the toothbrushes was revealed, six brushes were from HIV positive individuals and four were negative controls. One of the six toothbrushes from the HIV positive individuals was positive for HIV proviral DNA. This observation would be consistent with the clinical manifestation of hemorrhagic gingival in many HIV positive individuals. Ironically, a search of the literature revealed two separate articles where transmission of HIV from an infected brother to a non-infected brother occurred after sharing a toothbrush.

In addition, a case report was also published on an HIV positive patient who had Kaposi’s sarcoma of both the upper anterior gingival and the right palate. Initially, the patient presented with a CD4 cell count of 181 cells/mm3; a 3.5 cm x 2.6 cm x 2.3 cm deep purple lesion of the anterior maxillary gingival; and a 2.0 cm x 1.5 cm x >0.5 cm deep purple lesion sarcoma of the right posterior hard palate. Biopsy confirmed Kaposi’s sarcoma of both lesions. At the patient’s request, the only oral therapy that was instituted was that the patient was instructed to use two toothbrushes/day; placing each in the dishwasher after use, and changing both toothbrushes every week. After three months, the palatal lesion had increased in size by 1.0 cm in all dimensions while the anterior gingival lesion had decreased 0.5 cm in size in all dimensions. Glass et al suggested that the reason for the decrease in the anterior gingival lesion was due to the decreased microbial exposure to the lesion via the toothbrush.

In order to fulfill Koch’s postulates for disease transmission by the toothbrush, Glass et al chose the dog as the best animal model for toothbrush disease transmission study; in part because of the dog’s strong oral immune system (e.g., consider what dogs do with their mouths). The study employed a triple cross-over design where each dog received all three experimental treatments. Toothbrushes were initially sterilized using ethylene oxide. The three arms of the experimental design called for:
1) brushing each day for a month with a new sterile toothbrush each time; 2) brushing each day for a month with a sterile toothbrush that had been subsequently contaminated with a known-number of microorganisms; and 3) brushing each day for a month with the same toothbrush (sterilized prior to the initial brushing), simulating the daily brushing pattern of most humans. Local disease in the oral mucosa was scored and blood was drawn for blood cultures.

What this in vivo study showed was that daily-brushing, even with a new sterile toothbrush, can produce oral lesions if the “tooth-brusher” is not careful (design 1 above). It also showed that if the toothbrush is contaminated with a known pathogenic microorganism, it not only increases the number of intra-oral lesions, but the same microorganisms can be cultured from the bloodstream after brushing (design 2 above). However, the most striking finding of this study was that the repeated use of the same toothbrush had by far both more intra-oral lesions and blood-borne microorganisms (design 3 above), especially if the dogs were immune-suppressed. These findings are very similar to the clinical observations on toothbrushes of human subjects by other investigators.

One of the most compelling studies dealt with toothbrush end-rounding. Selected bristle tufts on new toothbrushes from three manufacturers were examined, using a dissecting microscope. The bristles were scored as to whether they were end-rounded or sharp. The toothbrushes were then used for one-week and two-week intervals. The same bristle tufts were again examined and scored. At the beginning of the study, all three toothbrush brands had 98-100% of the bristles rounded. After one week of use, 1/3 of the bristles from two of the toothbrush manufacturers were sharp and jagged; after two weeks of use, 2/3 of the bristles from the same two toothbrush manufacturers were sharp and jagged. The toothbrushes manufactured by the third company showed no change over the two week period; however, these bristles were very thick in terms of diameter. As part of this study though, bristles were not only photographed using the magnification provided by the dissecting microscope, they were also examined using a scanning electron microscope. When the bristles were examined under the scanning electron microscope, all three toothbrush brands showed rough and jagged edges. Similar studies were performed on eight toothbrush brands by Silverstone and Featherstone who actually found that end-rounding on unused brushes varied from 88% to 22% rounding/brush. Of course, the importance of end-rounding loss is that the sharp bristles can easily penetrate the mucosa, injecting the microorganism into the submucosal tissue, including the thin-walled vessels that reside there.

Presently, studies are being conducted on the most commonly used electric toothbrushes, and the preliminary data is somewhat alarming (Unpublished data). It appears that regardless of whether the toothbrush heads have a motorized movement or produce ultrasonic waves, large numbers of both pathogenic and opportunistic bacteria, yeasts, and even mold are housed in the heads of the brushes and then widely disseminated when the brushes are activated. Obviously, some of the food, water, and microorganisms are drawn back into the heads of the brushes during the act of brushing. These microorganisms proliferate when the brush is not being used. When the brush is subsequently turned on in the act of brushing, these microorganisms are thrust from the head/bristles into the mouth. Many of the microorganisms cultured from the electric and the ultrasonic toothbrushes are frank pathogens, including Aspergillus and other molds.

While the toothbrush problem was being delineated, studies were being conducted on a number of possible disinfection-techniques. The first techniques employed liquid agents: mouthrinses, Clorox, bourbon, scotch, vodka; even dishwashing and submersing the toothbrushes in super-saturated H2O2 overnight (Unpublished data). None of these was successful at reducing any more than the superficial colonies on the bristles. (Note: Earlier in vitro studies had shown that the microbial contamination was not only on the tops of the bristles, but throughout the entire length of the bristles and even the toothbrush head.) Attempts to sterilize the toothbrush were successful by both autoclaving and microwaving. However, by the time sterilization was reached, both methods caused the toothbrush heads to melt and the bristles to splay, making the sterile toothbrush non-functional. As mentioned earlier, toothbrushes can be sterilized without the distortion using an ethylene oxide sterilizer, but ethylene oxide is a known carcinogen and has an affinity for plastic. Such a procedure would not be a successful mechanism for everyday use in a household environment.

Additional unpublished studies looked at where toothbrushes were stored (eg., the bathroom) and found that the bathroom was the most contaminated room in the entire house. Because most of the population stored their toothbrushes in the bathroom, two studied were conducted to look at the use of toothbrush sanitizing devices. The first study looked at a U-V device manufactured by Associated Mill, Inc., in Chicago, IL. While this U-V device was effective at reducing some microorganisms, it acted as an incubator for others. Studies on the Purebrush Toothbrush Sanitizer (Murdock Laboratories, Burlingame, CA) showed that this unit effectively killed not only bacteria and yeasts/fungi, but also killed viruses.

With the research that has been conducted, it is clear that the toothbrush may become contaminated with both opportunistic and pathogenic microorganisms that are capable of producing both local (gingival, mucosal, and tongue) infections, but also systemic disease. The findings that the Purebrush Toothbrush Sanitizer was effective in eliminating such microorganisms from the toothbrush, gives rise to the following research questions:

1. Can the use of the Purebrush Toothbrush Sanitizer reduce the incidence of mucositis in children who are immune-suppressed by Acute Myelo-monocytic Leukemia and those awaiting transplantation?
2. Can the use of the Purebrush Toothbrush Sanitizer reduce the incidence of systemic diseases in children who are immune-suppressed by Acute Myelo-monocytic Leukemia (AML) and those awaiting transplantation?
3. What are the best clinical end-points for study in answering questions 1 and 2?


After having the study objectives and procedures explained to the parents or guardians, ten children with AML and ten children who are immune-suppressed awaiting organ transplant will be selected at the beginning of their immune-suppression therapy. Each child will have an initial oral and systemic health evaluation prior to beginning the study by a designated member of the Hematology/Oncology Clinical Service. Each child will then be given a Purebrush Toothbrush Sanitizer, six clear manual toothbrushes, that they will change every two weeks for the duration of the 12-week study and instructions on the use of the sanitization device. Chemotherapy will be conducted using the same protocol for all ten patients.

The children will be examined daily by their parents or guardians and a diary of the oral status and statements about the child’s general health will be recorded. Body temperatures will be obtained and recorded both in the morning upon awakening and in the evening just before retiring. Each subject will have both an oral and a systemic evaluation every three weeks while they are on chemotherapy by the original designated member of the Hematology/Oncology Clinical Service.


Since this is a pilot study, the data will be analyzed to determine appropriate end-points for additional larger studies. Special attention will be paid to oral end-points such as mucositis or the lack thereof and systemic end-points such as fever, diarrhea, and URI’s. The daily diaries will also be analyzed for un-expected trends in the study population.


•Glass RT, Lare MM. Toothbrush contamination: a potential health risk?
Quintessence International. 1986;17:39-42.
•Glass RT. Clinical Pathologic Conference: Other factors in infections: the transmission of disease.
Gerodontics. 1986;2:119-120.
•Glass RT, Jensen HG. More on the contaminated toothbrush: the viral story.
Quintessence International. 1988;19:713-716.
•Rubin E, Farber JL. Pathology, 3rd Ed. Philadelphia:
Lippincott-Raven 1999;368-370.
•Glass RT, Jensen H. The effectiveness of the pollenex ds60 daily dental sanitizer in reducing the number of bacteria, yeast and viruses on toothbrushes.
Okla Dental Assoc J. 1994;84:24-28.
•Glass RT, Carson SR, Barker RL, et al. Detection of HIV proviral DNA on toothbrushes: a preliminary study.
Okla Dental Assoc J. 1994;84: 17-20.
•HIV infection in two brothers receiving intravenous therapy for hemophilia.
MMWR. 1992;41:228-231.
•Fitzgibbon J, Gaur S, Frenkel L, et al. Transmission from one child to another of human immunodeficiency virus 1 with zidovudine-resistent mutation.
N. Engl J Med. 1993;325:1835-1841.
•Glass RT, Min K-W, Adler V. The toothbrush, kaposi’s sarcoma and aids: a case demonstrating interesting associations.
Okla Dent Assoc J, 1995;86:22-24.
•Smith AL. Microbiology and Pathology, 12Ed. St. Louis: C.V. Mosby 1980;97.
•Malmberg E, Birkhed D, Norvenius G, et al. Microorganisms on toothbrushes at day-care centers.
Acta Odontologica Scandinavica, 1994;52:93-98.
•Warren DP, Goldschmidt MC, Thompson MB, et al. The effects of toothpastes on the residual microbial contamination of toothbrushes.
JADA, 2001;132:1241-1245.
•Glass, R.T., Transmission of disease: the role of the toothbrush and the denture.
AAOPAbs., (63) April 25, 1990.
•Silverstone L, Featherstone M. Examination of end rounding pattern of toothbrush bristles using scanning electron microscopy: a comparison of eight toothbrush types.
Periodontics, 1988;445-62.
•Glass RT, Jensen HG. The effectiveness of a u-v toothbrush sanitizing device in reducing the number of bacteria, yeasts, and viruses on toothbrushes.
Okla Dent Assoc J, 1994;84:245-28.
•Glass R, Furgason M, Moody, J. In vivo study of the efficacy of an ultra-violet toothbrush sanitizer.
Abs. #2972, IADR, 1997.

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