Relationship Between the Presence or Absence of Gingival Bleeding and the Enzymatic BANA Test

Marcio Fernando de Moraes GRISI1
Tarcirio Alves CORREA FILHO2
Carmen Lucia Silva FANGANIELLO3
Walter MARTINS Jr.2
Cincinato Rodrigues SILVA-NETO1

1Faculty of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil
2Faculty of Dentistry, University of Ribeirão Preto (UNAERP), Ribeirão Preto, SP, Brazil
3School of Professional Training, Dental Association of Ribeirão Preto (AORP), Ribeirão Preto, SP, Brazil
4Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

Correspondence: Prof. Dr. Marcio Fernando de Moraes Grisi, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil. Tel: +55-16-602-3981. e-mail:

Braz Dent J (2001) 12(1): 23-26 ISSN 0103-6440


When periodontal disease started to be considered a bacterial infection mainly mediated by subgingival plaque, the basic problem faced by periodontists was the identification and/or quantification of periodontopathogenic bacteria. However, clinical methods continue to be of great value for the diagnosis of periodontal disease. In the present study we show a significant correlation between an index widely used in clinical practice, the Gingival Index of Löe (1967), based on the presence or absence of bleeding on probing, and the methodology of the BANA test for the detection of the specific enzymatic activity of microorganisms involved in periodontal disease. More sensitive and specific clinical parameters, taken together with other microbiologic methods, will be useful in daily clinical practice even before periodontal treatment.

Key words: bleeding on probing, BANA test, Gingival Index, periodontal disease


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. In 1967, Löe (1) used the presence or absence of gingival bleeding as one of the components of an index, and later other investigators used this clinical parameter as one of the major components of their respective indices (2-5).

The bleeding index has been used in a wide variety of studies (3,6,7), 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. (8), 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.

As previously demonstrated, periodontopathogenic bacteria produce enzymes (9) which have the ability to hydrolyze the synthetic substrate N-benzoyl-DL-argininenaphthylamide (BANA). The extent of BANA hydrolysis by subgingival plaque is significantly correlated with the number and proportions of spirochetes present in plaque samples from various sites (10) and at individual sites (11). In view of the fact that the BANA test has provided a perspective for bacteriologic monitoring during the different phases of periodontal treatment in clinical dentistry (12), the objective of the present study was to compare clinical evaluation of the gingival index (GI) (1) with the enzymatic BANA test.


Patients were selected at the Periodontic Clinic of the School of Professional Training of the Dental Association of Ribeirão Preto. Of the 28 patients selected for the study, none had received previous periodontal treatment. All patients were classified as having adult periodontitis and none of them had been using antibiotics or any other medication over the last six months. Also, none of them reported the presence of systemic disease. The GI of Löe (1) 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
Table 1. Frequency distribution of the gingival indices (GI) for 513 sites in 28 patients.

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 (1). 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 material was collected from these surfaces for analysis of BANA hydrolysis. Bacterial subgingival plaque samples were collected from 513 sites of 28 patients with the aid of a Gracey 5/6 periodontal curette. Supragingival plaque was not removed. GI analysis and collection of material were performed by a single professional before any procedure. To avoid contamination from one site to another when collecting material from the same patient, the periodontal probe and the curette were disinfected according to the criteria proposed by Murai et al. (13). The BANA hydrolysis test was performed as recommended by Loesche (9) and as described by Grisi et al. (12). The method used for the test was that of liquid form and the following scores were established: 1 = negative for yellow color; 2 = weekly positive for orange color; 3 = positive for reddish and intense red color. The mean GI and BANA test values were calculated for each tooth and surface for statistical analysis with the chi-square test, with the level of significance set at 0.05.


Table 1 presents the frequency of the GI of 513 sites in 28 patients. The highest percentages were for the GI 2 and GI 3 scores, 52.4% and 34.1%, respectively, which involve the presence of bleeding on probing as one of the major parameters.

Table 2 presents the distribution of GI and the intensity of the BANA test for the 513 sites analyzed during the initial examination of the patients, with significance determined by the chi-square test. The percentages of BANA score 1 decreased with increasing GI. For BANA score 2, percentages were similar. However, there was a direct relationship for BANA score 3, i.e., the percentage of sites tended to increase with increasing GI.

By pooling the GI 2 and GI 3 scores into a single group representing sites with (GI 2 + GI 3) and without (GI 1) bleeding, the new frequencies are reported in Table 3. BANA-positive and weakly positive cases predominated (86.6%) over negative cases (13.4%). The chi-square test applied to the total frequencies for the GI and BANA hydrolysis groups (Table 2) was significant at the 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.


Gingival bleeding is definitely an objective sign of inflammation of gingival connective tissue. It does not represent a diagnosis because it does not distinguish between the various forms of periodontal disease but it is associated with them. The occurrence of bleeding during brushing, when using dental floss or a toothpick, or during periodontal examination with a probe, is widely accepted by dentists as an objective sign of inflammatory periodontal disease and can be easily understood by patients. Bleeding is not only an unequivocal and objective sign of gingivitis or of different manifestations of periodontitis, but also precedes other objective signs such as changes in color and edema.

A periodontal probe has been used by several researchers (4,5) to elicit bleeding, although a triangular dental toothpick (3,14) or even dental floss (2) have also been used. Although there are studies that define gingival bleeding as a limited and inefficient clinical sign of moderate sensitivity for the evaluation of periodontal disease and for the monitoring of loss of insertion (15-17), others consider it to be a consistent and sensitive clinical parameter (18).

When this clinical parameter was correlated with the analysis of microorganisms in the subgingival plaque (the objective of the present study), a relevant result was added to clinical evaluation, i.e., the presence of periodontogenic microorganisms. In the present study, a correlation between the GI and positive results of
BANA hydrolysis was clearly established (Table 3). The ability of subgingival plaque to hydrolyze BANA has been significantly associated with untreated periodontal disease (9).

Considering that periodontal disease is of bacterial origin, it is imperative to find clinical parameters that may be easily utilized in daily practice in an objective and safe manner and that will be positively correlated with the extent of periodontal infection. It was for this reason that, in order to verify the degree of reliability of bleeding on probing for the evaluation of gingival conditions by the GI of Löe (1), we used the enzymatic method of BANA hydrolysis, which has been widely reported in the literature as a reliable measurement of the extent of periodontal infection.

The method for the enzymatic BANA test used in the present study was the liquid phase one. Although we recognize that this method may be difficult to apply in clinical dentistry, its use is of high value for research, as shown by the countless reports (19) in the literature since the technique was first used in 1982 (20). Starting in 1989, thanks to the scientific evidence obtained with the liquid phase BANA method, the first studies using the solid phase or the card (Perioscan) were carried out (8). In a comparative study of the two methodologies, both techniques were found to be reliable (8), with the card form clearly proving to be easy to use by clinicians, and with the results being obtained at the same visit as the collection of samples.

The present results obtained using the enzymatic BANA test in comparison with the presence or absence of bleeding on probing, analyzed by the gingival index, proved to be of great value as a diagnostic aid in periodontal disease, reinforcing the clinical evaluations made during the phase of initial patient examination. This test will definitely help clinicians and periodontists in terms of prevention, for a more effective evaluation of treatment and for patient monitoring during supportive periodontal therapy visits.


Grisi MFM, Correa Filho TA, Fanganiello CLS, Martins Jr W, Silva-Neto CR, Salvador SL. Relação entre a presença ou ausência de sangramento gengival e o teste enzimático de BANA. Braz Dent J 2001;12(1):23-26.

Quando a doença periodontal foi considerada como uma infecção bacteriana, mediada pela placa subgingival, o problema básico para o periodontista foi a identificação e/ou quantificação das bactérias periodontopatogênicas. Entretanto os critérios clínicos continuam sendo de grande valia para o diagnóstico das doenças periodontais. No presente estudo, foi encontrado uma correlação significante entre um índice largamente utilizado na prática clínica, o Índice Gingival de Löe (1967), baseado na presença ou ausência de sangramento à sondagem, e o teste BANA, utilizado para diagnosticar a presença de infecção anaeróbia, através da produção da enzima arginina hidrolase. Exames clínicos específicos, usados em conjunto com métodos microbiológicos, podem ser úteis na clínica, mesmo antes de se iniciar o tratamento periodontal.

Unitermos: sangramento à sondagem, teste BANA, Índice Gengival de Löe, doênça periodontal.


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Accepted June 4, 2000
Braz Dent J 12(1) 2001