Year : 2006 | Volume
: 17 | Issue : 1 | Page : 2--6
Detection and measurement of oral malodour in periodontitis patients
Mathew John, KL Vandana
Department of Periodontics, College of Dental Sciences, Davangere-577 004, Karnataka State, India
K L Vandana
Department of Periodontics, College of Dental Sciences, Davangere-577 004, Karnataka State
BACKGROUND & OBJECTIVES : Malodour has been correlated with the concentration of volatile sulphur compounds produced in the oral cavity by metabolic activity of bacteria colonizing the periodontal sites and the dorsum of the tongue. The aim of this study was to detect malodour in mouth air organoleptically and using a portable sulphide monitor and to correlate it with the clinical parameters, halitosis linked toxins and BANA, using tongue and subgingival plaque samples. The halitosis grading is also correlated with the microbial colonies of the subgingival plaque sample. METHODS : 20 patients with chronic periodontitis with 5-7 mm pocket depth, radiographic evidence of bone loss and presence of oral malodour participated in this study. Assessment of mouth air was done organoleptically and by using a portable sulphide monitor. The clinical parameter, plaque index (PI), gingival index (GI), gingival bleeding index (BI), were obtained from all the areas. Samples for BANA and to detect halitosis linked toxins were taken from the dorsal surface of the tongue and periodontal pockets ranging 5-7 mm. Halitosis related microbial colonies were identified using anaerobic culturing from the subgingival plaque. RESULTS : The scores of PI, GI, BI and sample that tested positive for halitosis linked toxins and with the halitosis grading were not significant. The presence of tongue coating and the halitosis grading and toxin levels were significant. BANA has shown to be non contributory due to technical problems. Anaerobic culture has shown to identify Streptococcus, Bacteroides, Fusobacterium, Porphyromonas and Prevotella colonies. INTERPRETATION & CONCLUSION : The results confirmed that there was no correlation between the clinical parameters, halitosis linked toxins and halitosis grading. The microbial colonies have shown to correlate with the presence of oral malodour.
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John M, Vandana K L. Detection and measurement of oral malodour in periodontitis patients.Indian J Dent Res 2006;17:2-6
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John M, Vandana K L. Detection and measurement of oral malodour in periodontitis patients. Indian J Dent Res [serial online] 2006 [cited 2017 Feb 28 ];17:2-6
Available from: http://www.ijdr.in/text.asp?2006/17/1/2/29899
Oral malodour (also known as bad breath, halitosis, fetor ex ore) usually originates when the halitosis emitted through the mouth combines with malodourous compounds originating in the oral cavity. Bad breath has a significant impact on our daily social life to those who suffer from it. Oral malodour may rank only behind dental caries and periodontal disease as the cause of patient's visit to the dentist. Surprisingly, enough, until recently breath malodour has not been a matter of much interest in periodontology, even though it is of such high priority to the public. This malodour causing volatile sulphur compounds result from microbial fermentation of proteins, peptides and mucins found in saliva, blood, postnasal drip, gingival crevicular fluid, lysed neutrophils and desquamated epithelial cells . Many studies have demonstrated that hydrogen sulphide (HS), methyl mercaptan (CH,SH) and to a lesser extent, dimethyl sulphide (CH,SCH) accounts 90% of the total volatile sulphur compounds (V SCs) found in the mouth air, suggesting that these volatile sulphur compounds arethe chemicals responsible for halitosis .
Among the gram-negative bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Treponema denticola, the so-called periodontopathogens are major contributors of volatile sulphur compounds ,,. Subjects with periodontal disease frequently suffer from oral malodour and positive correlation has been demonstrated between severity of periodontitis and volatile sulphur compound (VSC levels . Thus the microbiological analysis of these organisms responsible for volatile sulphur compound production and toxins confirms the association of halitosis with periodontitis.
So far, the periodontal literature tries to focus oral malodour either with clinical parameters and / or culture techniques. Till date, the study of oral malodour using clinical parameters, a rapid chair side tests like halitosis linked toxin reagents and culture technique have not been included together in the Indian scenario. The purpose of this study is to correlate halitosis grading in chronic periodontitis patients to periodontal clinical parameters, halitosis linked toxins using Halitox using both tongue and subgingival plaque samples. Also, the halitosis grading is correlated with the microbial colonies of the subgingival plaque samples alone.
MATERIALS AND METHODS
The patients for this study were selected from the Department of Periodontics, College of Dental Sciences, Davangere, Karnataka. A total of 20 patients from both sexes were included in the study.
Patients suffering from chronic periodontitis with 5-7 mm pocket depth, radiographic evidence of boneloss and presence of oral malodour were included in this study. Subjects taking antibiotics within the last 3 months, who have undergone any periodontal therapy for the past 6 months, patients suffering from any systemic disease (e.g. diabetes mellitus, chronic renal failure, cirrhosis of liver, gastrointestinal disorder, respiratory dysfunction and various carcinoma etc), which are known to cause oral malodour and female patients who are pregnant and lactating were excluded from this study.
The selected patients underwent recording of the clinical parameters: Plaque Index (Silness and Loe, 1964), Gingival Index (Loe and Silness, 1963), Gingival Bleeding Index (Ainamo and Bay, 1975) and Pocket depth measurement using an UNC- 15 probe.
The probing pocket depth was measured with UNC-15 periodontal probe. The probe was inserted parallel, to the long axis of the tooth gently till resistance was noted and readings were recorded to the nearest millimeter. Organoleptic examination, considered as a reference standard for oral malodour detection was done. To detect the odour from the mouth the patient was asked to pinch the nose with the finger, stop his or her breathing for a moment with the lips sealed and then exhaled gently by opening the mouth. The odour detected this waywas from local factors of the oropharyngeal cavity .
Recording of malodour (halitosis) was done using a small hand held breath checking device, which detected the volatile sulphur compounds and hydrocarbon gases in mouth air. As the monitor was turned on, it emitted a beep, when a second beep was heard the patient was asked to blow into the air flow passage. After the third beep, odour levels were measured by one of the gradings on the graphic display of the instrument (i.e. no odour, slight odour, moderate odour, strong odour). If no number appeared then it was considered a reading error and the procedure was repeated. The organoleptic analysis (nasal sniffing) was used to select the patients which was confirmed by the small portable sulphide monitor (Tanita°) [Figure 1]. No attempt was made to quantify the volatile sulphur compounds levels in the patients.
Halitosis linked toxins was measured using Halitox", a quick, simple colorimetric test that detects both volatile sulphur compounds such as hydrogen sulphide and methyl mercaptan as well as polyamines like putrescine and cadaverine. Using sterile cotton tipped applicator the tongue sample was taken from the dorsum of the tongue and using sterile curette, pooled subgingival plaque sample was obtained and placed on the cotton applicator. After the sample were obtained the cotton tip was quickly submersed in the reaction tubes separately and thehandle was broken off at the top of the vial. The screw was then replaced and the reaction was allowed to proceed for a total of 2 minutes. After 2 minutes, the sample tube was held against a color chart for interpretation. The color chart contains 3 color scale-low toxin, moderate toxin and high toxin [Figure 3].
BANA' Reagent test designed as a simple colorimetric test for the detection of enzymes in tongue coating and subgingival plaque sample that hydrolyses the synthetic peptide N-Benzoyl-DL-Arginine-b-Naphthylamide (BANA). These enzymes, produced by three anaerobic periodontopathic species, includes Porphyromonas gingivalis, Bacteroides forsythus and Treponema denticola, that in vitro produce copious amount of malodomous compounds [Figure 2].
Using a sterile curette, the pooled subgingival plaque samples were collected from various pockets of 5-7 mra pocket depth and were placed on a glass slide and using a cotton tip swab, the coating from the posterior dorsum of the tongue was also removed. The pocket and tongue samples were placed on the lower reagents strip separately, folded and incubated for 15 minutes at 55 f5°sub C.
Bacterial sampling for anaerobic culture, known to be the 'gold standard' technique was used in this study. Subgingival plaque samples were taken from the selected pockets by means of sterile curette which was placed into 4m1 thyoglycolate broth with heroin and vitamin K (transporting media) and sealed tightly to avoid contamination.
The samples were sent to anaerobic culturing lab within 24-48 hours. 10 microlitre was taken and placed on 3 culture media, i.e blood agar, brewer anaerobic agar and Bacteroides Bile Esulin. The sample was incubated for 72 hours at37°sub C. After culturing the samples were placedon a glass slide. The smear was made and was fixed by heat fixation. Gram staining was done and was observed under the stereomicroscopeto identify the microbial colonies.
All the descriptive data that included mean, standard deviation and percentage frequency were determined for each categories. Categorical data were analysed either by Chi-square or Fisher's Exact test. Continuous data (clinical parameters) were compared by Mann-Whitney test. Relationship was assessed by Spearman's correlation coefficient (halitosis grading and clinical parameters).
[Table 1] shows the subject demographic factors (age and sex). The result confirmed in [table 2] shows that with the increase in the halitosis grading and pocket toxin scoring, there was an increase in the plaque index, gingival index, bleeding index and pocket depth. Further toxin scoring of the tongue and pocket sample demonstrated medium score in 55% and 70% respectively and high score was seen in 35% and 30% respectively [Table 3].
In [Table 4], the microbial examination showed that the percentage of Streptococcus colonies and the Bacteroides colonies increased with the increasing grades of halitosis. The results in [Table 5] shows that there was no significant correlation between the halitosis grading and toxin scorings. Table 6 presents the microbial profile and the pocket toxin score. The results of this study showed that there was 28.6% Streptococcus colonies, 57.1% Fusobacterilrn colonies, 14.3% Porphyromonas colonies for the halitosis grading of 2. In grade 3, 50% Streptococcus colonies, 20% Fusobacterium colonies, 10% Porphyromonas, Prevotella and Bacteroides colonies and only 66.6% Streptococcus colonies for the halitosis grading of 4. The BANA reagent test results have failed to show any results in this study.
Previous studies revealed sufficient information to determine that the major cause of bad breath is oral microflorathat produces volatile odouriferous molecules, including sulphur compounds and organic acids among others ,. Oral malodour may provide a window for diagnosis of periodontal disease. Recently a small hand held monitor (Tanita°'. Japan) was introduced for the measurement of volatile sulphur compound in mouth air . The portable monitor provided a subjective reading which favours the organoleptic assessment. The simple and compact detectors have been developed employing semi conductor gas sensors and these are becoming popular in the clinical field.
The nasal sniffing  in this study was used to detect the presence or absence of malodour, as the organoleptic measurement by multiple judges is cumbersome and introduces problems of subj ectivity and reproducibility.
The organoleptic analysis (nasal sniffing) was used to select the patients which was confirmed by the small portable sulphide monitor (Tanita°'). No attempt was made to quantify the volatile sulphur compounds levels in the patients.
In this study, the mean plaque score has shown to increase for the halitosis grading of 2 to 4 which is not significant, similartotheobservation by BosyAetal, andcontrast to Yaegaki et al , . For the pocket depth range of 5-7 mm various grades of halitosis has been recorded from low to high, which was not significant in accordance with the findings of BosyA et al ,  and Morita M et al ,  and in contrast to Yaegaki K et al , .
The mean bleeding percentage increased for the halitosis grading of 2 to 4 with a maximum score (82.3%) seen for the halitosis grading of 4, however the mean bleeding score was not significant in contrast to the findings of Tonzetich J et al , , Yaegaki K et al ,  and MoritaM et al , .
For the halitosis grading of 2 to 4, the mean gingival score was not significant, in contrast to the observation made by Kostel JG et al , . The results of this study showed, that the toxins have shown to have a correlation with the tongue coating sample and subgingival pocket plaque sample. As no studies exist inthe literature using Halitox~ to measure toxins, the results of this study could not be correlated.
In this study, the tongue plaque sample was not considered for anaerobic culture for the following reasons; very few data exist on the type of bacteria present on the tongue surface of people with subjective complaints of oral malodour. There are large variability in contrast of individual species. This finding is consistent with the previous studies of the tongue flora, which showed similar fluctuations in the total count and in the prevalence and proportion of recovered individual species. The variability may be explained in parts by the difficulty to obtain a standardize sample of the tongue flora .
Till date the subgingival pocket-microbial colonies have not been studied to correlate with the halitosis grading. In this study an attempt was made to isolate Streptococcus, Bacteroides, Porphyromonas, Prevotella, Fusobacterium colonies from the subgingival plaque.
The Streptococcus colonies seem to predominate in various grades followed by Fusobacterium, Porphyromonas, Prevotella and Bacteroides colonies. The presence of these organism were found to correlate with the increasing halitosis grading. The results of this study showed, that the toxins have shown to have a correlation with the tongue coating sample and subgingivalpocket plaque sample.
Since there is pancity of literature using Halitox to measure toxins, we could not correlate our results.
Due to technical problems, the BANA reagent test was not able to demonstrate any results in this study. The earlier studies conducted by Vandana KL and Kala M, 1997  and Vandana KL and Apama B 1998  utilized the BANA (BANA Scan Oral B, USA) effectively without any technical and obvious reasons. The BANA test kit is an expensive advanced chairside kit, which identifies BANA positive organisms that are also main odouriferous periodontal microorganism was tried in this study. The BANA test kit used to acid an important microbial information was of futile effort. However several studies report significant association of BANA positive organisms i.e. Porphyromonas gingivalis and Bacteroides forsyrthus to be associated with the oral malodour,.
The conclusions drawn from this study are; the detection of oral malodour in chronic periodontitis patients was done organoleptically. The oral malodour was of moderate to severe grades as detected by using a portable sulphide monitor (Tanita). The malodour (halitosis) grading was correlated with the periodontal clinical parameters such as plaque index, gingival index, gingival bleeding index and pocket depth measurements, but no significant correlation was obtained. No correlation was obtained also on comparison between oral malodour and halitosis linked toxins. The beneficial effect of BANAwas not established as BANA test did not show any results. A correlation was seen between the microbial colonies and different oral malodour gradings. There was varied presence of Streptococcus, Fusobacterium, Prevotella, Bacteroides and Porphymmonas colonies with different grades of malodour.
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