Oral Diseases (2013) doi:10.1111/odi.12210 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd All rights reserved www.wiley.com
ORIGINAL ARTICLE
The relationship between tongue brushing and halitosis in children: a randomized controlled trial T Ileri Keceli1, D Gulmez2, A Dolgun3, M Tekcicek1 1
Department of Paediatric Dentistry, Faculty of Dentistry, Hacettepe University, Ankara, Turkey; 2Department of Medical Microbiology, Faculty of Medicine, Hacettepe University, Ankara, Turkey; 3Department of Biostatistics, Faculty of Medicine, Hacettepe University, Ankara, Turkey
OBJECTIVE: The study aimed to investigate the clinical and microbiological effects of tongue brushing on malodour in children. SUBJECTS AND METHODS: One hundred and fiftyone caries-free children were included. After clinical evaluation, halitosis was determined by organoleptic assessment and sulphide monitoring. Then, 69 children with high levels of volatile sulphur compounds (VSC) were randomly assigned into two groups (group 1: scaling-polishing + tooth brushing + tongue brushing and group 2: scaling-polishing + tooth brushing), and tongue coating samples were collected for microbiological analysis. After 2 weeks, VSC measurements, organoleptic assessment, clinical evaluations and sample collection were repeated. RESULTS: In both groups, organoleptic scores, VSC levels, gingival index, plaque index (PI), bleeding on probing and Winkel tongue coating index (WTCI) scores decreased after 15 days. However, only the change in WTCI and PI scores showed a statistically significant intergroup difference. The most prevalent anaerobic bacteria were Veillonella spp., Prevotella spp., Fusobacterium spp., and no intergroup difference was observed in terms of colony counts of bacteria. CONCLUSIONS: Tongue brushing did not provide an additional benefit to the treatment for malodour. According to the microbiological culture results, a specific bacterium responsible for halitosis in children could not be identified and more sensitive methods might be used for this purpose. Oral Diseases (2013) doi:10.1111/odi.12210 Keywords: halitosis; children; tongue brushing; microbiology
Correspondence: Tulin Ileri Keceli, Department of Paediatric Dentistry, Faculty of Dentistry, Hacettepe University, 06100 Sihhiye-Ankara/ Turkey. Tel: +90 312 3052280, Fax: +90 312 3243190, E-mail: tulinileri@ yahoo.com Received 1 October 2013; revised 2 November 2013; accepted 6 November 2013
Introduction ‘Halitosis’ is a term used to identify offending breath originating from both non-oral and oral sources (Cortelli et al, 2008). The aetiology of halitosis is multifactorial depending upon systemic and oral conditions, but among all cases, 80–90% is related to intraoral conditions (Miyazaki et al, 1995; Delanghe et al, 1997). Intraoral halitosis originates from bacterial putrefaction of organic substrates especially existing in oral soft tissues, saliva, epithelial cells, crevicular fluid and food debris (McNamara et al, 1972; Tonzetich, 1977; Persson et al, 1989, 1990; Kleinberg and Westbay, 1992) and is called oral malodour. The main putrefaction products are volatile sulphur compounds (VSC), and major components of VSC are methyl mercaptan (CH3SH), dimethyl sulphide [(CH3)2S] and hydrogen sulphide (H2S) (Tonzetich, 1977; Schmidt et al, 1978; Yaegaki and Sanada, 1992b). Microorganisms producing VSC involve proteolytic anaerobes, mostly gram-negative species, which are primarily located in tongue coating and periodontal pockets (Tonzetich, 1977; Yaegaki and Sanada, 1992a; Donaldson et al, 2005; Washio et al, 2005). The rough surface morphology over the dorsum of the tongue with fissures and crypts provides a large ecological oral site supporting the debris accumulation and the development of an anaerobic microbiota for the production of malodorous products (Jacobson et al, 1973; Bosy et al, 1994). Therefore, tongue coating is known as a main source of intraoral halitosis (De Boever and Loesche, 1995; Rosenberg, 1996; Nakano et al, 2002). In many studies involving adults, the relationship between tongue coating and halitosis was demonstrated (Yaegaki and Sanada, 1992a; Miyazaki et al, 1995; Quirynen et al, 1998; Morita and Wang, 2001; Oho et al, 2001). Besides, it was reported that a reduction was observed in bacterial load on the tongue and a significant decrease in halitosis was achieved following the application of tongue cleaning (Gilmore and Bhaskar, 1972; Gilmore et al, 1973; De Boever and Loesche, 1995; Quirynen et al, 2004; Van der Sleen et al, 2010). However, a few studies reported in the literature
Tongue brushing and halitosis in children
T Ileri Keceli et al
2
investigated the relationship between oral health parameters and halitosis in children (Amir et al, 1999; Lin et al, 2003; Kara et al, 2006; Nalcaci and Sonmez, 2008; Nalcaci et al, 2008). Moreover, there is only one study evaluating the intraoral bacterial population of children with halitosis (Paryavi-Gholami et al, 1999), and to our knowledge, no controlled clinical trial investigating the effect of tongue brushing on oral malodour in child population was reported. From this point of view, the hypothesis to be tested was that in children, oral health parameters are related to halitosis and tongue cleaning reduces halitosis. The aim of our two-phase, single-blind, randomized, controlled clinical study was to investigate the factors affecting intraoral halitosis and determine the clinical and microbiological effects of tongue brushing on oral malodour in children.
Materials and methods Study population and study design The study was designed as a randomized, single-centred, single-blind (observer) controlled clinical trial and conducted at the Hacettepe University Faculty of Dentistry, Department of Paediatric Dentistry from 2009 to 2011. All subjects were selected from 613 patients who visited our clinic for various complaints. The purpose and possible complications were explained to the participants and their parents, and they were kindly asked to sign an informed consent. An approval was obtained from the institutional ethical committee (FON 08/12-53), and the experimental protocol was carried out according to the principles manifested in the Declaration of Helsinki and consistent with good clinical practices. Patient inclusion criteria were as follows: (i) ages between five and twelve; (ii) no space maintainers, orthodontic appliances or retainers; (iii) no systemic disease (including renal disease, respiratory tract diseases, diabetes mellitus, gastrointestinal tract disorders, acute or chronic sinusitis, kidney disorders, liver disorders); (iv) no pathology of the oral tissues (pericoronitis, dental/periodontal abscess, etc.); (v) no active caries lesions; (vi) no fissured tongue, geographic tongue; (vii) no gagging reflex preventing tongue sampling or treatment procedures; (viii) no habitual mouth breathing; (ix) no extraoral halitosis; (x) no medication and systemic antibiotic therapy in the preceding 4 weeks; (xi) no usage of anti-microbial mouthwashes. In the first part of the study, demographic information, medical history, dental history and oral hygiene habits were retrieved. Then, clinical evaluation and oral malodour assessment were performed. Clinical evaluation Oral health condition of the patients was established using decayed, missing and filled teeth (DMFT, DMFS, dmft, dmfs) indices (Baelum et al, 1997), gingival index (GI) (Silness and Loe, 1964), plaque index (PI) (Silness and Loe, 1964), bleeding on probing (BOP) (Badersten et al, 1984), Winkel tongue coating index (WTCI) (Winkel et al, 2003), presence of food impaction, salivary pH (Ericsson, 1953), salivary flow rate (Ericsson, 1953) and salivary buffering capacity (Ericsson, 1953). Oral Diseases
Halitosis assessment Halitosis was evaluated by organoleptic assessment and sulphide monitoring using a Halimeter (Interscan, Chatsworth, CA, USA). Subjects were given oral and written instructions regarding food intake and oral hygiene practices before halitosis assessment. They were instructed (i) to avoid eating food containing onions, garlic, spices and using mouthwashes 48 h before assessments and (ii) to refrain from drinking, eating, brushing, tongue cleaning and chewing for 2 h before each visit (Donaldson et al, 2007). Hour intervals between 9 and 11 a.m. were selected for measurement. Organoleptic assessment Organoleptic assessment was made before all other measurements by a single calibrated examiner who had been previously tested for smell acuity using the Smell Identification Test â (Sensonics Inc., Haddon Heights, NJ, USA). The examiner avoided drinking tea, coffee, using scented cosmetics and smoking before organoleptic assessment (Yaegaki and Coil, 2000). Exhaled air from the nose was evaluated to rule out extraoral halitosis. Halitosis was scored according to the scale of Rosenberg and McCulloch, which has a grading between 0 and 5 (Rosenberg and McCulloch, 1992). With regard to the scale, halitosis is graded as unperceivable (score 0), barely noticeable (score 1), slight but clearly noticeable (score 2), moderate (score 3), strong (score 4) and extremely strong (score 5). Organoleptic assessments were performed before VSC measurements. Sulphide monitoring (VSC measurements) As a primary outcome measure, total VSC levels in breath were detected using Halimeter (Interscan). The VSC levels ≥ 150 peak parts per billion (ppb) or organoleptic scores ≥ 2 were recorded as halitosis, while the levels lower than these indicated the absence of halitosis (Dal Rio et al, 2007; Brunner et al, 2010). Measurements were repeated three times consecutively, and the average ppb value of VSC level was recorded for each subject. Treatment procedures To assess the clinical and microbiological effect of tongue brushing, 69 volunteer children with a level of VSC ≥ 150 ppb and an organoleptic score of two or more were involved in the second part of the study (Figure 1). These children were randomly assigned with a sealed envelope design into two groups with respect to treatment procedure by one of the authors (M.T.). Group 1: routine oral care procedures including scaling-polishing were employed and the patients were instructed to make tooth brushing and tongue cleaning twice a day, after breakfast and before bedtime. Group 2: routine oral care procedures including scaling-polishing were employed and the patients were instructed to brush their teeth twice a day, after breakfast and before bedtime. In both groups, the children and their parents were instructed about tooth brushing and tongue cleaning by the same investigator (M.T.) in accordance with the treatment procedures. For tongue brushing procedure, subjects were demonstrated to (Danser et al, 2003) (i) pull out the tongue as far as possible, (ii) place the
Tongue brushing and halitosis in children
T Ileri Keceli et al
3
Figure 1 Subject flow diagram of the study.
toothbrush as far posterior as possible to the dorsum of the tongue, (iii) pull the toothbrush forward slowly to the front of the mouth, (iv) remove the debris from the toothbrush under running water and (v) repeat the scraping procedures until further debris cannot be removed. For microbiological evaluation, in this session, tongue coating samples were collected from the posterior dorsal surface of the tongue. After 2 weeks, VSC measurements, organoleptic assessment, tongue coating sample collection and clinical evaluations were repeated. Under the same conditions, all clinical measurements, VSC measurements and organoleptic assessments were taken by the same investigator (T.I.K.) who was blinded to the treatment procedure. Microbiological sampling and evaluation of the tongue For anaerobic culture, 1 cm2 of tongue surface was gently scraped six times with a sterile spatula and the scrapings were placed in 1 ml of thioglycolate medium. Samples were transported to the laboratory as soon as possible. In the laboratory, samples were vortexed vigorously for 30 s and diluted 100 times in saline, and 10 ll of the dilutions was cultured on blood agar, Schaedler agar, MacConkey agar and chocolate agar. While blood agar, chocolate agar and MacConkey agar were incubated in 5% CO2 at 36 2°C overnight, Schaedler agar was incubated at 36 2°C anaerobically by using GasPak EZ Anaerobe Gas Generating Container System (Becton Dickinson, Sparks, MD, USA) and evaluated after 2 and 4 days of incubation. Aerobic growth of alpha-haemolytic streptococci, coagulase-negative staphylococci and Neisseria spp. was quantitated. Growth other than normal oral flora such as gram-negative enteric bacilli, beta-haemolytic streptococci, Streptococcus pneumoniae and Haemophilus spp.
was also checked. Colony morphology, Gram staining, catalase (3% H2O2), oxidase and coagulase tests were used for the identification of aerobic bacteria. Aerotolerance test was performed from all macroscopically different colonies grown on Schaedler agar. Anaerobic colonies were quantified and identified by Gram staining, pigment formation, catalase (15% H2O2) and indole tests and Crystal system (Becton Dickinson). Data were log10-transformed for statistical analysis. Statistical analysis For all statistical analyses, SPSS for Windows (version 15.0, 2006 SPSS Inc., Chicago, IL, USA) program was used. Normality of the data was checked by using Kolmogorov–Smirnov test. ANOVA test was performed to clarify the relationship between VSC measurements and organoleptic scores. In halitosis-positive and negative subjects, clinical parameters were compared by using chi-square test. In addition, dependent t-test and independent t-test were performed for comparing intragroup and intergroup changes. Significance level was declared at P < 0.05 for all statistical evaluations. In order to evaluate the interobserver reliability and degree of concordance for the organoleptic and clinical measurements (DMFT, DMFS, dmft, dmfs indices, PI, GI, BOP, WTCI), the Pearson and Spearman correlation coefficient (for quantitative measures) and the Kappa statistic (for qualitative measures) were used. Group sample sizes of 34 and 35 were found as adequate to achieve 99% power for 2.0 difference between groups with 0.05 significance level following a two-sided two-sample t-test. Oral Diseases
Tongue brushing and halitosis in children
T Ileri Keceli et al
4
values ≥ 150 ppb and score 2 or more with organoleptic assessment (halitosis + (Figure 1)). VSC levels were significantly associated with organoleptic scores (P < 0.05). Significant correlations were detected between halitosis and dmft, dmfs indices, GI, PI, BOP and WTCI (P < 0.05). However, DMFT, DMFS indices, salivary pH, salivary flow rate and salivary buffering capacity were not correlated with halitosis (P > 0.05) (Table 1). Interobserver reliability was high for all organoleptic and clinical measurements. For organoleptic assessments, the interobserver reliability was j = 0.76 P < 0.001, whereas all correlation coefficients were r = 1 (all P < 0.001) for DMFT, DMFS, dmft and dmfs; P < 0.001 r = 0.999 for GI, PI and BOP; and P < 0.001 r = 0.988 for WTCI.
Table 1 The relationship between clinical measurements and halitosis in children with and without halitosis
Salivary pH Salivary flow rate Salivary buffering capacity DMFT DMFS dmft dmfs PI GI BOP WTCI
Halitosis ( ) mean s.d.
Halitosis (+) mean s.d.
P
7.684 0.318 0.833 0.323 4.465 0.929
7.699 0.254 0.897 0.330 4.535 0.995
0.756 0.229 0.652
0.489 0.509 0.016* 0.004*
ORIGINAL ARTICLE
The relationship between tongue brushing and halitosis in children: a randomized controlled trial T Ileri Keceli1, D Gulmez2, A Dolgun3, M Tekcicek1 1
Department of Paediatric Dentistry, Faculty of Dentistry, Hacettepe University, Ankara, Turkey; 2Department of Medical Microbiology, Faculty of Medicine, Hacettepe University, Ankara, Turkey; 3Department of Biostatistics, Faculty of Medicine, Hacettepe University, Ankara, Turkey
OBJECTIVE: The study aimed to investigate the clinical and microbiological effects of tongue brushing on malodour in children. SUBJECTS AND METHODS: One hundred and fiftyone caries-free children were included. After clinical evaluation, halitosis was determined by organoleptic assessment and sulphide monitoring. Then, 69 children with high levels of volatile sulphur compounds (VSC) were randomly assigned into two groups (group 1: scaling-polishing + tooth brushing + tongue brushing and group 2: scaling-polishing + tooth brushing), and tongue coating samples were collected for microbiological analysis. After 2 weeks, VSC measurements, organoleptic assessment, clinical evaluations and sample collection were repeated. RESULTS: In both groups, organoleptic scores, VSC levels, gingival index, plaque index (PI), bleeding on probing and Winkel tongue coating index (WTCI) scores decreased after 15 days. However, only the change in WTCI and PI scores showed a statistically significant intergroup difference. The most prevalent anaerobic bacteria were Veillonella spp., Prevotella spp., Fusobacterium spp., and no intergroup difference was observed in terms of colony counts of bacteria. CONCLUSIONS: Tongue brushing did not provide an additional benefit to the treatment for malodour. According to the microbiological culture results, a specific bacterium responsible for halitosis in children could not be identified and more sensitive methods might be used for this purpose. Oral Diseases (2013) doi:10.1111/odi.12210 Keywords: halitosis; children; tongue brushing; microbiology
Correspondence: Tulin Ileri Keceli, Department of Paediatric Dentistry, Faculty of Dentistry, Hacettepe University, 06100 Sihhiye-Ankara/ Turkey. Tel: +90 312 3052280, Fax: +90 312 3243190, E-mail: tulinileri@ yahoo.com Received 1 October 2013; revised 2 November 2013; accepted 6 November 2013
Introduction ‘Halitosis’ is a term used to identify offending breath originating from both non-oral and oral sources (Cortelli et al, 2008). The aetiology of halitosis is multifactorial depending upon systemic and oral conditions, but among all cases, 80–90% is related to intraoral conditions (Miyazaki et al, 1995; Delanghe et al, 1997). Intraoral halitosis originates from bacterial putrefaction of organic substrates especially existing in oral soft tissues, saliva, epithelial cells, crevicular fluid and food debris (McNamara et al, 1972; Tonzetich, 1977; Persson et al, 1989, 1990; Kleinberg and Westbay, 1992) and is called oral malodour. The main putrefaction products are volatile sulphur compounds (VSC), and major components of VSC are methyl mercaptan (CH3SH), dimethyl sulphide [(CH3)2S] and hydrogen sulphide (H2S) (Tonzetich, 1977; Schmidt et al, 1978; Yaegaki and Sanada, 1992b). Microorganisms producing VSC involve proteolytic anaerobes, mostly gram-negative species, which are primarily located in tongue coating and periodontal pockets (Tonzetich, 1977; Yaegaki and Sanada, 1992a; Donaldson et al, 2005; Washio et al, 2005). The rough surface morphology over the dorsum of the tongue with fissures and crypts provides a large ecological oral site supporting the debris accumulation and the development of an anaerobic microbiota for the production of malodorous products (Jacobson et al, 1973; Bosy et al, 1994). Therefore, tongue coating is known as a main source of intraoral halitosis (De Boever and Loesche, 1995; Rosenberg, 1996; Nakano et al, 2002). In many studies involving adults, the relationship between tongue coating and halitosis was demonstrated (Yaegaki and Sanada, 1992a; Miyazaki et al, 1995; Quirynen et al, 1998; Morita and Wang, 2001; Oho et al, 2001). Besides, it was reported that a reduction was observed in bacterial load on the tongue and a significant decrease in halitosis was achieved following the application of tongue cleaning (Gilmore and Bhaskar, 1972; Gilmore et al, 1973; De Boever and Loesche, 1995; Quirynen et al, 2004; Van der Sleen et al, 2010). However, a few studies reported in the literature
Tongue brushing and halitosis in children
T Ileri Keceli et al
2
investigated the relationship between oral health parameters and halitosis in children (Amir et al, 1999; Lin et al, 2003; Kara et al, 2006; Nalcaci and Sonmez, 2008; Nalcaci et al, 2008). Moreover, there is only one study evaluating the intraoral bacterial population of children with halitosis (Paryavi-Gholami et al, 1999), and to our knowledge, no controlled clinical trial investigating the effect of tongue brushing on oral malodour in child population was reported. From this point of view, the hypothesis to be tested was that in children, oral health parameters are related to halitosis and tongue cleaning reduces halitosis. The aim of our two-phase, single-blind, randomized, controlled clinical study was to investigate the factors affecting intraoral halitosis and determine the clinical and microbiological effects of tongue brushing on oral malodour in children.
Materials and methods Study population and study design The study was designed as a randomized, single-centred, single-blind (observer) controlled clinical trial and conducted at the Hacettepe University Faculty of Dentistry, Department of Paediatric Dentistry from 2009 to 2011. All subjects were selected from 613 patients who visited our clinic for various complaints. The purpose and possible complications were explained to the participants and their parents, and they were kindly asked to sign an informed consent. An approval was obtained from the institutional ethical committee (FON 08/12-53), and the experimental protocol was carried out according to the principles manifested in the Declaration of Helsinki and consistent with good clinical practices. Patient inclusion criteria were as follows: (i) ages between five and twelve; (ii) no space maintainers, orthodontic appliances or retainers; (iii) no systemic disease (including renal disease, respiratory tract diseases, diabetes mellitus, gastrointestinal tract disorders, acute or chronic sinusitis, kidney disorders, liver disorders); (iv) no pathology of the oral tissues (pericoronitis, dental/periodontal abscess, etc.); (v) no active caries lesions; (vi) no fissured tongue, geographic tongue; (vii) no gagging reflex preventing tongue sampling or treatment procedures; (viii) no habitual mouth breathing; (ix) no extraoral halitosis; (x) no medication and systemic antibiotic therapy in the preceding 4 weeks; (xi) no usage of anti-microbial mouthwashes. In the first part of the study, demographic information, medical history, dental history and oral hygiene habits were retrieved. Then, clinical evaluation and oral malodour assessment were performed. Clinical evaluation Oral health condition of the patients was established using decayed, missing and filled teeth (DMFT, DMFS, dmft, dmfs) indices (Baelum et al, 1997), gingival index (GI) (Silness and Loe, 1964), plaque index (PI) (Silness and Loe, 1964), bleeding on probing (BOP) (Badersten et al, 1984), Winkel tongue coating index (WTCI) (Winkel et al, 2003), presence of food impaction, salivary pH (Ericsson, 1953), salivary flow rate (Ericsson, 1953) and salivary buffering capacity (Ericsson, 1953). Oral Diseases
Halitosis assessment Halitosis was evaluated by organoleptic assessment and sulphide monitoring using a Halimeter (Interscan, Chatsworth, CA, USA). Subjects were given oral and written instructions regarding food intake and oral hygiene practices before halitosis assessment. They were instructed (i) to avoid eating food containing onions, garlic, spices and using mouthwashes 48 h before assessments and (ii) to refrain from drinking, eating, brushing, tongue cleaning and chewing for 2 h before each visit (Donaldson et al, 2007). Hour intervals between 9 and 11 a.m. were selected for measurement. Organoleptic assessment Organoleptic assessment was made before all other measurements by a single calibrated examiner who had been previously tested for smell acuity using the Smell Identification Test â (Sensonics Inc., Haddon Heights, NJ, USA). The examiner avoided drinking tea, coffee, using scented cosmetics and smoking before organoleptic assessment (Yaegaki and Coil, 2000). Exhaled air from the nose was evaluated to rule out extraoral halitosis. Halitosis was scored according to the scale of Rosenberg and McCulloch, which has a grading between 0 and 5 (Rosenberg and McCulloch, 1992). With regard to the scale, halitosis is graded as unperceivable (score 0), barely noticeable (score 1), slight but clearly noticeable (score 2), moderate (score 3), strong (score 4) and extremely strong (score 5). Organoleptic assessments were performed before VSC measurements. Sulphide monitoring (VSC measurements) As a primary outcome measure, total VSC levels in breath were detected using Halimeter (Interscan). The VSC levels ≥ 150 peak parts per billion (ppb) or organoleptic scores ≥ 2 were recorded as halitosis, while the levels lower than these indicated the absence of halitosis (Dal Rio et al, 2007; Brunner et al, 2010). Measurements were repeated three times consecutively, and the average ppb value of VSC level was recorded for each subject. Treatment procedures To assess the clinical and microbiological effect of tongue brushing, 69 volunteer children with a level of VSC ≥ 150 ppb and an organoleptic score of two or more were involved in the second part of the study (Figure 1). These children were randomly assigned with a sealed envelope design into two groups with respect to treatment procedure by one of the authors (M.T.). Group 1: routine oral care procedures including scaling-polishing were employed and the patients were instructed to make tooth brushing and tongue cleaning twice a day, after breakfast and before bedtime. Group 2: routine oral care procedures including scaling-polishing were employed and the patients were instructed to brush their teeth twice a day, after breakfast and before bedtime. In both groups, the children and their parents were instructed about tooth brushing and tongue cleaning by the same investigator (M.T.) in accordance with the treatment procedures. For tongue brushing procedure, subjects were demonstrated to (Danser et al, 2003) (i) pull out the tongue as far as possible, (ii) place the
Tongue brushing and halitosis in children
T Ileri Keceli et al
3
Figure 1 Subject flow diagram of the study.
toothbrush as far posterior as possible to the dorsum of the tongue, (iii) pull the toothbrush forward slowly to the front of the mouth, (iv) remove the debris from the toothbrush under running water and (v) repeat the scraping procedures until further debris cannot be removed. For microbiological evaluation, in this session, tongue coating samples were collected from the posterior dorsal surface of the tongue. After 2 weeks, VSC measurements, organoleptic assessment, tongue coating sample collection and clinical evaluations were repeated. Under the same conditions, all clinical measurements, VSC measurements and organoleptic assessments were taken by the same investigator (T.I.K.) who was blinded to the treatment procedure. Microbiological sampling and evaluation of the tongue For anaerobic culture, 1 cm2 of tongue surface was gently scraped six times with a sterile spatula and the scrapings were placed in 1 ml of thioglycolate medium. Samples were transported to the laboratory as soon as possible. In the laboratory, samples were vortexed vigorously for 30 s and diluted 100 times in saline, and 10 ll of the dilutions was cultured on blood agar, Schaedler agar, MacConkey agar and chocolate agar. While blood agar, chocolate agar and MacConkey agar were incubated in 5% CO2 at 36 2°C overnight, Schaedler agar was incubated at 36 2°C anaerobically by using GasPak EZ Anaerobe Gas Generating Container System (Becton Dickinson, Sparks, MD, USA) and evaluated after 2 and 4 days of incubation. Aerobic growth of alpha-haemolytic streptococci, coagulase-negative staphylococci and Neisseria spp. was quantitated. Growth other than normal oral flora such as gram-negative enteric bacilli, beta-haemolytic streptococci, Streptococcus pneumoniae and Haemophilus spp.
was also checked. Colony morphology, Gram staining, catalase (3% H2O2), oxidase and coagulase tests were used for the identification of aerobic bacteria. Aerotolerance test was performed from all macroscopically different colonies grown on Schaedler agar. Anaerobic colonies were quantified and identified by Gram staining, pigment formation, catalase (15% H2O2) and indole tests and Crystal system (Becton Dickinson). Data were log10-transformed for statistical analysis. Statistical analysis For all statistical analyses, SPSS for Windows (version 15.0, 2006 SPSS Inc., Chicago, IL, USA) program was used. Normality of the data was checked by using Kolmogorov–Smirnov test. ANOVA test was performed to clarify the relationship between VSC measurements and organoleptic scores. In halitosis-positive and negative subjects, clinical parameters were compared by using chi-square test. In addition, dependent t-test and independent t-test were performed for comparing intragroup and intergroup changes. Significance level was declared at P < 0.05 for all statistical evaluations. In order to evaluate the interobserver reliability and degree of concordance for the organoleptic and clinical measurements (DMFT, DMFS, dmft, dmfs indices, PI, GI, BOP, WTCI), the Pearson and Spearman correlation coefficient (for quantitative measures) and the Kappa statistic (for qualitative measures) were used. Group sample sizes of 34 and 35 were found as adequate to achieve 99% power for 2.0 difference between groups with 0.05 significance level following a two-sided two-sample t-test. Oral Diseases
Tongue brushing and halitosis in children
T Ileri Keceli et al
4
values ≥ 150 ppb and score 2 or more with organoleptic assessment (halitosis + (Figure 1)). VSC levels were significantly associated with organoleptic scores (P < 0.05). Significant correlations were detected between halitosis and dmft, dmfs indices, GI, PI, BOP and WTCI (P < 0.05). However, DMFT, DMFS indices, salivary pH, salivary flow rate and salivary buffering capacity were not correlated with halitosis (P > 0.05) (Table 1). Interobserver reliability was high for all organoleptic and clinical measurements. For organoleptic assessments, the interobserver reliability was j = 0.76 P < 0.001, whereas all correlation coefficients were r = 1 (all P < 0.001) for DMFT, DMFS, dmft and dmfs; P < 0.001 r = 0.999 for GI, PI and BOP; and P < 0.001 r = 0.988 for WTCI.
Table 1 The relationship between clinical measurements and halitosis in children with and without halitosis
Salivary pH Salivary flow rate Salivary buffering capacity DMFT DMFS dmft dmfs PI GI BOP WTCI
Halitosis ( ) mean s.d.
Halitosis (+) mean s.d.
P
7.684 0.318 0.833 0.323 4.465 0.929
7.699 0.254 0.897 0.330 4.535 0.995
0.756 0.229 0.652
0.489 0.509 0.016* 0.004*
