Relationship between gingival bleeding and anaerobic periodontal infection assessed by BANA (N-Benzoyl-DL-Arginine-β-Napthylamide) assay.

BACKGROUND: Since periodontal diseases are primarily inflammatory in nature, the ability to detect inflammatory lesions in gingival tissue is essential for the diagnosis and monitoring of changes in gingival status. Gingival bleeding has been used as a reliable and objective clinical parameter not only for the evaluation of inflammatory conditions of periodontium, but also in the detection of its activity and progression. The gingival bleeding index (Loe) is based on the presence or absence of gingival bleeding, thereby provides a means of converting observed clinical data into statistical analysis. Periodontitis is widely regarded as a bacterial infection mainly mediated by subgingival plaque. N-benzoyl-DL-arginine β-naphthylamide (BANA) test is a simple chair side test used to detect, the specific enzymatic activity of microorganisms involved in periodontal disease.
OBJECTIVE: The objective of this study is to compare the clinical evaluation of the gingival index (GI) with the enzymatic BANA test.
MATERIALS AND METHODS: A total of 320 sites were selected from 20 patients with generalized chronic periodontitis, based on the presence are absence of gingival bleeding according to GI of Loe. Sub-gingival plaque samples were collected using a sterile curette and the BANA test was performed.
RESULTS: The distribution of GI and the intensity of the BANA test for the 320 sites analyzed during the initial examination of patients, with significance determined by the Chi-square test. The results were statistically significant at P ≤ 0.000.
CONCLUSION: There was a direct relationship between positive BANA test results and GI scores, clearly demonstrating the validity of gingival bleeding as a significant diagnostic parameter. CLINICAL SIGNIFICANCE: The validity of gingival bleeding as a clinical tool not only for the diagnosis of periodontal disease, but also in the detection of its activity and progression could be established by correlating the GI scores (which is based on the presence or absence of bleeding) with the primary etiologic factors (putative periodontal pathogens) assessed by BANA assay.

Periodontal disease comprises of a group of inflammatory conditions of the supporting tissues of the teeth, which is characterized by loss of connective tissue attachment and alveolar bone resulting in the formation of probable pockets around the teeth. Since periodontal diseases are primarily inflammatory in nature, the ability to detect inflammatory lesions in gingival tissues is essential for the diagnosis and monitoring of changes in gingival status. Gingival bleeding has been widely utilized as a reliable, safe, and objective clinical parameter for the evaluation of the inflammatory conditions of gingival connective tissues. Clinical indices provide a means of converting observed clinical data into numerical data for statistical analysis.[1] The gingival bleeding index (Loe) has been used in a wide variety of studies,[2] demonstrating the interest of this clinical parameter for investigators in the evaluation of periodontal conditions, not only in terms of the diagnosis of periodontal disease, but also in the detection of its activity and progression. However, a diagnosis based solely on clinical criteria may be considered unreliable because, according to Loesche et al.,[3] a simple bacteriological test would be useful in office practice in order to identify the presence or prevalence of microorganisms used as indicators of periodontal disease.

The primary etiological agent in chronic periodontitis is plaque microorganisms. As previously demonstrated, periodontopathogenic bacteria produce enzymes,[4] which have the ability to hydrolyze the synthetic substrate N-benzoyl-DL-arginine β-naphthylamide (BANA). BANA test is a simple chair side test, which detects the putative microorganisms present in plaque, which is widely reported in the literature as a reliable measurement of the extent of anaerobic periodontal infection. The extent of BANA hydrolysis by sub-gingival plaque is correlated with bleeding on probing, which is used to evaluate the gingival conditions by the gingival index (GI) of Loe, to verify the validity of GI. The objective of this study was to compare clinical evaluation of the GI with the enzymatic BANA test.

Materials and Methods

The subjects for this study were selected from the patients attending a private clinic in Chennai, India. The patients were selected by a convenient sampling technique based on clinical examination using the following criteria.[5] Patients suffering from generalized form of chronic periodontitis, above the age group of 35 years, free from systemic diseases, patients who have not received any local or systemic antimicrobial and anti-inflammatory therapy for the past 6 months, patients who have not received any periodontal treatment for at least 1 year before examination, pregnant women and women who were on contraceptive medication were not selected for the study. The GI of Löe was analyzed in order to determine the extent of inflammation of periodontal tissues, with emphasis on the dichotomous analysis of the presence or absence of bleeding on probing. GI was determined using a periodontal probe with millimeter divisions in order to verify the presence or absence of bleeding on probing, and clinical analyses according to the criteria described by Löe. GI was classified as follows: GI 1, mild inflammation without bleeding on probing and only a slight alteration in color and edema; GI 2, moderate inflammation with redness, edema, smooth surface, and bleeding on probing; GI 3, severe inflammation, clearly visible redness, edema, ulceration, and a tendency to spontaneous hemorrhaging.

The evaluations were performed in the proximal (mesial and distal) areas of the teeth and the material was collected from these surfaces for analysis of BANA hydrolysis from 20 patients with generalized form of chronic periodontitis (more than 30% of sites with probing depth more than 5 mm, or attachment loss more than 1 mm). The BANA test detects the synthesis of a trypsin-like enzyme produced by three putative periodontal pathogens: Porphyromonas gingivalis, Bacteroides forsythus and Treponema denticola. This enzyme hydrolyzes the synthetic peptide of the BANA reagent and releases a chromophore, beta-napthylamide, which is coupled to Evans black dye, and read as a color change on the reagent test card.

According to the test protocol, supragingival plaque was removed and sub-gingival plaque samples were collected with curettes, placed on reagent cards, incubated for 15 min at 55°C and visually evaluated for color changes and the results were interpreted using the BANA reagent interpretation chart.

Negative: No blue color is observable on a pale red back ground

Weak positive: Faint blue color on a pale red background

Positive: Distinct patches of blue somewhat larger and darker than the weak positive reaction on a pale red background.

The results so obtained were subjected to statistical analysis and plotted in the form of tables.

Results

Table 1 presents the frequency of GI for 320 sites in 20 patients (the most affected sites in each sextants were considered). The highest percentages were for the GI 2 and GI 3 scores, 20 and 63.8%, respectively, which involve the presence of bleeding on probing as one of the major parameters

Table 1

Frequency distribution of GI for 320 sites

Table 2 presents the distribution of GI and the results of the BANA test for the 320 sites analyzed during the initial examination of patients, with significance determined by the Chi-square test. The percentage of BANA score 1 decreased with increasing GI. For BANA score 2, percentage were gradually increased with increasing GI. However, there was a direct relationship for BANA score 3, that is, the percentage of sites tended to increase with increasing GI [Figure 1].

Table 2

Distribution of GI and of intensity of the BANA test for 320 sites at first examination

Figure 1

Distribution of gingival index and of intensity of the N-benzoyl- DL-arginine β-naphthylamide test for 320 sites at first examination

By pooling GI 2 and GI 3 into a single group representing sites with bleeding (GI 2 + GI 3) and without (GI 1) bleeding, the frequencies are reported in Table 3 [Figure 2]. BANA positive and weak positive cases predominated (83.71%) over negative (16.25%) cases. The Chi-square test applied to the total frequencies for the GI and BANA hydrolysis group [Table 2] was significant at 1% level of probability. When GI with bleeding was regrouped for comparison with GI without bleeding [Table 3], the Chi-square test revealed a nonsignificant value for score 1 BANA hydrolysis, but a significant value when scores 2 and 3 of BANA hydrolysis were compared.

Table 3

Frequency distribution of sites with and without bleeding on probing and BANA hydrolysis

Figure 2

Frequency distribution of sites with and without bleeding on probing and N-benzoyl-DL-arginine β-naphthylamide hydrolysis

Discussion

The objective of the present study was to determine the validity of gingival bleeding as a definitive clinical parameter for the evaluation of inflammatory conditions of the gingival. Since periodontal diseases are mainly inflammatory in nature, the ability to detect inflammatory lesions in gingival tissue, forms the basis for the diagnosis and monitoring of changes in periodontal status.[1] Gingival bleeding apart from indicating the presence of inflammation, also serves as indicator for sites of disease activity, although it does not represent a diagnosis as it cannot be used to differentiate between the various forms of periodontal disease, but usually associated with them.[6] These findings clearly demonstrate the significance of using gingival bleeding to detect the presence of periodontal disease. Hence, the GI (Loe 1967) was used in this study as it uses the presence or absence of gingival bleeding as one of its major components.

The primary etiologic factors in periodontal disease are plaque bacteria. Although several distinct bacterial species are present in the oral cavity, a limited number of them are found to be strikingly related to clinical measurements of periodontal disease (viz. P. gingivalis, Tanerella forsythia, T. denticola, and other spirochetes).[7] BANA test (BANAMET LLC) format was used because it is helpful as a simple objective chair side test to find out the presence of putative periodontal pathogens; however, it does not identify which of the three BANA positive species is present in the plaque, but as all the three species are anaerobes it allows the clinician to determine the presence of an anaerobic periodontal infection,[8] hence, it was used in the present study.

The validity of gingival bleeding as a clinical tool not only for diagnosis of periodontal disease, but also in its activity and progression could be established by correlating the GI scores (which is based on presence or absence of bleeding) with the primary etiologic factors (putative periodontal pathogens) assessed by BANA assay, hence this study was performed.

In this study, 320 sites were selected from 20 patients according to the scoring criteria for GI and the BANA test was performed. When the GI scores were correlated with the BANA test results out of the 320 sites examined, 133 sites (41.6%) were weak positive and 137 sites (42.8%) were positive which was in accordance with Loesche,[9] Muthukumar and Diviya.[7]

GI score 1 showed significant correlations with negative BANA reactions (64%). The presence of positive (2.2%) may be due to the presence of preclinical infection (presence of microorganisms, but not to the extent of producing disease).[10] The highly significant positive BANA test reactions for GI scores 2 and 3 suggest a significant correlation between increasing GI scores and putative periodontal pathogens. These findings are in agreement with the results of Syed et al. (1984),[11] Grisi et al. (2011), Muthukumar et al. (2010 and 2013). The negative BANA test reactions may be due to the presence of putative microorganisms below the detection level of BANA test and/or presence of nonputative microorganisms.

Conclusion

The results clearly demonstrates the direct relationship between increasing GI scores and positive BANA test results, which clearly demonstrates the significance of gingival bleeding in the diagnosis of periodontal disease, thereby clearly demonstrating the validity of GI.