19

Summary

Among the paraclinical methods that can be used for an accurate assessment of the periodontalstatus prior and during the periodontal treatment we present the importance of using two tests,the biochemical TOPAS test and the enzymatic BANA test. Both tests have a good practical impor-tance proven by statistical good correlation between the results of TOPAS and the papilla bleedingindex, and BANA tests and the clinical form of periodontal disease.The conclusion of the study encourages the use of such chair-side tests for a proper diagnostic ofperiodontal disease and for a good evaluation of the treatment results.

Keywords: gingivitis, topas test, periodontits, bana test.

Introduction

It is known that periodontal disease is pro-duced by the bacteria residing in the oralbiofilm, named also bacterial plaque. It isdifficult to isolate and grow many of thosebacterial species by the culture methodbecause this requests good equipment andeven though, some of the bacterial speciesare uncultivable [1]. The DNA probes andpolymerase chain reaction (PCR) techniquescan detect even the uncultivable bacteria[2,3], but they require good laboratoryequipment and therefore are not used as rou-tine examination in the dental surgery.

In 1992, dr. Löesche introduced a chair-side quick microbial-enzymatic test, BANA,in order to assess the presence of some bac-terial pathogen species in the subgingivalplaque [4].

Recent studies have shown that the pro-gression of periodontal destruction due tobacterial pathogen species is episodic anddoes not occur in a continuous, linear man-ner. Furthermore, periodontal pockets can

be active or inactive in the disease process[5]. Conventional diagnosis indicators, suchas probing depth, attachment level, bleedingon probing, gingival redness, gingivalindex, and assessment of alveolar bone onradiographs, cannot distinguish betweendisease activity and inactivity. Additionaldiagnosis information is therefore needed.

The analysis of crevicular fluid [6]is one possibility, because its componentsshow the etiopathogenical phenomena pro-duced in the periodontium, being consideredas markers of the progression and severityof periodontal disease. This can be done bya biochemical test named TOPAS (TOxicityPrescreening ASsay), which detects theindirect presence of bacteria by two markersof gingival infection: bacterial toxins andbacterial proteins. This test can be associat-ed with the severity of inflammation andwith the evolution of a destructive process,making the difference between an active andan inactive periodontal disease. We havedescribed the practical importance ofTOPAS test in a previous work. This work

Biochemical and enzymatic diagnosis aids in periodontal disease

Cristina Gabriela Puºcaºu1, Anca Dumitriu2, Horia Traian Dumitriu2

1 Constanta, 2 Bucharest, Romania

2. The principle of TOPAS test is thedetection of the presence of actively grow-ing and dividing anaerobic pathogens whichresults in increased levels of their toxicmetabolites in the crevicular fluid. The peri-odontal pathogens are producing continu-

ously toxic metabolites [9] and some of thencan be found in the crevicular fluid. Theyare also releasing bacterial proteins andendotoxins, which also can be found in thecrevicular fluid [10]. The host subjected tobacterial aggression releases certain factors

follows the previous research and its aim isto check if there exists a statistical positivecorrelation of those tests with clinical find-ings and with the severity of periodontalinvolvement.

Materials and Method

1. The principle of BANA test is to detectthe presence of three anaerobic bacteriaassociated with periodontal disease by theanalysis of subgingival plaque. These threebacteria are: Porphyromonas gingivalis,Treponema denticola, and Bacteroidesforsythus. Socransky and Haffajee (1997)concluded that these three pathogens have agreat prevalence in patients with periodonti-tis [7]. BANA test can detect the presence ofthese three microorganisms associated withperiodontal disease but it cannot make thedifference between them [8]. The BANApositive bacterial species contain a certainenzyme that can hydrolyze the peptideimpregnated on BANA strips. This peptideis N-benzoyl-DL-arginine-B-napthylamide

(BANA). When samples containing any ofthe three bacteria are placed on a BANA-impregnated test strip, a hydrolytic reactiongives the strip a distinctive blue color. Thedarker the blue, the more organisms arepresent.

The test (Figure 1) consists of a plasticstrip to which two separate reagent matricesare attached. The lower white reagent matrixis impregnated with N-benzoyl-DL-argi-nine-B-napthylamide (BANA). Subgingivalplaque samples are applied to the lowermatrix. The upper buff reagent matrix con-tains a chromogenic reagent that reacts withone of the hydrolytic products of theenzyme reaction to form a blue color. Theupper matrix should be moistened withsaline solution after sampling. Then theplastic strip is folded (so the two matricescome in contact) and placed for 5 minutes inincubator, which is set at 55 °C. The bluecolor appears in the upper buff matrix and ispermanent; the intensity of the color deter-mines whether it is a positive or weak posi-tive reaction.

OHDMBSC - Vol. IV - No. 4 - December, 2005

20

Figure 1. The BANA recipient containing 20 test strips

Figure 2. TOPAS kit containing 10 vials with eachreagent, yellow for bacterial toxins and blue for bac-terial proteins

of the immune response: antibodies,cytokines, which are responsible for theinduction of inflammatory response in peri-odontal structures [11].

The greater the degree of the metabolicactivity, the higher the concentrations ofthese toxins, shown by the color intensityscale of the TOPAS test. This test includestwo reagents, one for measuring the concen-tration of bacterial toxins and the second formeasuring the level of total proteins in thecrevicular fluid (Figure 2). First the paperpoint imbedded in crevicular fluid is intro-duced in the yellow cap vial. Here the reac-tion of the bacterial toxins from the crevicu-lar fluid with certain chemical colorlessreagents contained in the yellow cap vialtakes place. At the end of the reaction (5minutes) the color will turn to yellow. Thegreater the concentration of bacterial toxinsin the sample, the brighter yellow color willresult from the reaction. The second stepconsists in measuring the level of total pro-teins (including antibodies, human serumalbumin, aspartate aminotransferase, betaglucuronidase and bacterial proteins).Bacterial infection has been shown toincrease the levels of these and other pro-teins in crevicular fluid. The second reac-tion of TOPAS is based on the color changeproduced by adding the reagent from theblue cap vial. The greater the level of totalprotein in the GCF sample, the brighter theassay mixture will turn to blue.

Based on the above-mentioned theoret-ical considerations we assessed the clinicaland therapeutic importance of these twotests on a group of 61 subjects, all with dif-ferent forms of dental crowding and gingivi-tis or periodontitis. The subjects are young,aged between 15 and 46 years of age.Clinical chart was done in all patientsincluding periodontal examination beforeand after treatment. A number of 32 patientsfollowed orthodontic treatment and the restonly periodontal treatment consisting indebridement and application of local anti-

septic substances. BANA test was done afterorthodontic treatment in case of 32 patientsand after periodontal debridement in the restof 29 patients. TOPAS test was done in allpatients twice: first before any treatment andthe second time after orthodontic alignmentor after scaling and root planning in peri-odontal treated group of patients. The pro-gram used for the statistical assessment ofthis case control study is MedCalc®, ver-sion 7.3.0.1 [12]. The results were analyzedusing Spearman`s rank correlation coeffi-cient.

The purpose of the study is to establishif there exists any correlation between theclinical periodontal findings and the para-clinical results of TOPAS and BANA tests.

Results

The variables used for the statistical studyare: The Plaque Index (PI), The PapillaBleeding Index (PBI), the severity of crowd-ing assessed by TSALD coefficient, level ofbacterial toxins (BT) measured with TOPAStest, level of total proteins (TP) measuredalso with TOPAS test and the concentrationof pathogen anaerobic bacteria evaluatedwith BANA test as negative, weak positiveor positive.

We present a 31-year-old patient,female, with chronic marginal profoundperiodontitis (adult periodontitis).

Before treatment, TOPAS test is per-formed. The paper point is inserted in theperiodontal pocket (Figure 3) and left oneminute to absorb the fluid. The result(Figure 4) is a moderate bacterial toxicity(degree 3 on the scale).

Then the content of the blue cap vial isadded and after 2 minutes the color turned toblue. The result is an intermediate concen-tration of the total proteins in the pocketfluid (Figure 5).

Those results suggest a moderate levelof gingival inflammation in an evolutivestate.

OHDMBSC - Vol. IV - No. 4 - December, 2005

21

The subgingival plaque was removedwith a Gracey curette from the lower frontalarea, where the periodontal involvement ismore severe and placed on the lower matrix

of the BANA strip (Figure 7).After the strip was incubated for 5 min-

utes at 55-Celsius degrees we obtained aweak positive result in all 4 samples (Figure6). This result in a patient with objectivesigns of periodontal inflammation shows thepresence of periodontal pathogen bacterialspecies and requires a systemic antibiother-apy.

Because on the TOPAS scale the levelof total proteins is evaluated in letters fromA to D we associated to each letter a numberfrom 1 to 4 in order to ease the statisticalcalculation. The same was done for theBANA results, associating the value 1instead of negative result, 2 instead of weak-ly positive and 3 instead of positive BANAresult.

OHDMBSC - Vol. IV - No. 4 - December, 2005

22

Figure 5. The level of proteins in the pocket fluidis intermediate

Figure 3. The crevicular fluid is absorbed with thepaper point

Figure 4. The result of TOPAS indicate a moderatebacterial toxicity

Figure 6. BANA test is weak positive Figure 7. The BANA test strip

In Table 1 we observe that in our studythere is no statistical correlation between theseverity of crowding and the degree ofinflammation assessed with PBI index aswell as between severity of crowding andTOPAS results. There is a statistical goodcorrelation (almost perfect) between PBIand the level of bacterial toxins (BT), shownby rho index close to value 1 (which is theideal situation) and the probability that the

null hypothesis (P) is tending to zero. If P islower than 0.05 it is considered that there isenough evidence of the correctitude of theresults (to reject the null hypothesis). A goodcorrelation was found also between thedegree of gingival inflammation (PBI) andthe level of total proteins (TP) measuredwith TOPAS.

The results after treatment (periodontaland orthodontic) are presented in Table 2.

The same positive correlation betweenthe severity of bleeding and TOPAS find-ings are found after treatment, when bothvalues (PBI index and concentration of bac-terial toxins) decreased and we deal with animprovement of the periodontal condition,shown clinically by the reduction of thebleeding tendency and paraclinically by thereduction of the level of toxicity in thecrevicular fluid, measured with TOPAS test.

After testing BANA in all patients we

compared the results with the severity ofperiodontal involvement evaluated by thetype of disease. The patients included in thisstudy were diagnosed with one of the fol-lowing disease: simple chronic gingivitiswith microbial origin (was attributed thevalue 1 for the statistic calculation), margin-al chronic superficial periodontitis (wasnoted with value 2) and chronic marginalprofound (adult) periodontitis (was notedwith value 3).

There is a statistical positive correlationbetween the results of BANA test and thedegree of periodontal involvement, meaning

gingivitis, superficial periodontitis and pro-found periodontitis assessed with Spearmancoefficient (Table 3).

OHDMBSC - Vol. IV - No. 4 - December, 2005

23

PBI TOPAS TP TOPAS BT

rho P rho P rho P

Severity ofcrowding -0.275 0.0332 0.086 0.5037 -0.174 0.1785

PBI 1 < 0.00001 0.345 0.0075 0.635 < 0.0001

Table 1. Correlation between PBI and TOPAS findings before treatment (Spearman�s rank correlation coefficient)

PBI TOPAS TP TOPAS BTrho P rho P rho P

PBI 1 < 0.00001 0.495 0.0001 0.547 < 0.0001

Table 2. Correlation between PBI and TOPAS findings after treatment(Spearman�s rank correlation coefficient)

CF PIrho P rho P

BANA 0.752 < 0.00001 0.325 0.0118

Table 3. Correlation between BANA results and periodontal disease and plaque

Discussion

In all patients the periodontal debridement(associated or not with orthodontic align-ment) led to a reduction of clinical signs ofinflammation and a reduction of the bacteri-al toxicity level in the crevicular fluidassessed with TOPAS test. A statistical goodcorrelation was found between the degree ofgingival inflammation assessed with bleed-ing index (PBI) and the level of bacterialtoxicity assessed by TOPAS test.

Most patients having gingivitis hadBANA negative results, suggesting that thethree periodontal pathogens (Treponemadenticola, Porphyromonas gingivalis andBacteroides forsythus) are absent or exist inlow concentrations (less than 10.000colonies forming units in the sample of sub-gingival plaque). A weak positive BANAresult was obtained in certain patients withlong-evolution gingivitis. These patients areincluded in a risk category and without aproper therapy they can develop superficialperiodontitis and further adult periodontitis.

In most patients exhibiting superficialperiodontitis the BANA results are weakpositive, even negative in few cases. A weakpositive BANA result in a patient thatexhibits periodontal inflammatory signsshows a high risk to develop irreversibleperiodontal destruction. This fact is accord-

ing to the prognosis and evolution ofuntreated superficial periodontitis. Inpatients with profound periodontitis (adultperiodontitis) BANA results were positiveor weak positive. This finding means thatthe periodontal destruction in most of casesis accompanied by the presence in subgingi-val plaque of the BANA positive anaerobicspecies Treponema denticola, Porphyromonasgingivalis and Bacteroides forsythus.Therefore, systemic antibiotherapy isrequired in such cases of BANA positivepatients.

Conclusion

We conclude that both paraclinical examsTOPAS and BANA are quick and easy to beperformed in the dental surgery. Theyproved good correlation with the degree ofgingival inflammation and severity of theperiodontal disease. Therefore, these testshelp the clinician in elaborating a correctdiagnosis based on current evaluation ofperiodontal status and to elaborate a com-plex periodontal treatment based on clinicaland paraclinical findings. This study wishesto encourage the clinicians to use such chair-side tests for the diagnosis of periodontaldisease.

References

1. Sanz M, Lau L, Herrera D, Morillo JM, Silva A.Methods of detection of Actinobacillus actino-mycetemcomitans, Porphyromonas gingivalis, andTannerella forsythensis in periodontal microbiology,with special emphasis on advanced molecular tech-niques: a review. J Clin Periodontol 2004; 31: 1034-1047.

2. Jervøe-Storm P-M, Koltzscher M, Falk W, DorflerA, Jepsen S. Comparison of culture and real-time PCRfor detection and quantification of five putative peri-odontopathogenic bacteria in subgingival plaque sam-ples. J Clin Periodontol 2005; 32: 778-783.

3. Socransky SS, Haffajee AD. Periodontal micro-bial ecology. Periodontology 2000, 2005; 38: 135-187.

4. Löesche WJ: Comparison of the benzoyl-DL-argi-nine-naphthylamide (BANA) test, DNA probes, andimmunological reagents for ability to detect anaerobicperiodontal infections due to Porphyromonas gingi-valis, Treponema denticola and Bacteroides forsythus.J. Clin Microbiol. 1992; 30: 427-433

5. Shimada K, Mizuno T, Ohshio K, Kamaga M,Murai S, Ito K. Analysis of aspartate aminotransferasein gingival crevicular fluid assessed by usingPocketWatchTM: a longitudinal study with initialtherapy. J Clin Periodontol 2000; 72: 819-823.

6. Armitage GC. Analysis of gingival crevice fluid

OHDMBSC - Vol. IV - No. 4 - December, 2005

24

and risk of progression of periodontits,Periodontology 2000, 2004; 34: 109-119.

7. Socransky SS, Haffajee AD, Cuglini MA.Microbial complexes in subgingival plaque. J DentRes 1997; 76(special issue): abst 302.

8. Ellen RP, Galimanas VB. Spirochetes at the fore-front of periodontal infections. Periodontology 2000,2005; 38: 13-32.

9. Dumitriu HT, Dumitriu S. Periodontology. (inRomanian) Ed. Viaþa Medicalã Româneascã 1999; 63-65, 72-74.

10. Lindhe J, Karring T, Lang NP. ClinicalPeriodontology and Implant Dentistry, BlackwellMunksgaard 2003: 164-166, 211-213.

11. Vernal R, Dutzan N, Chaparro A, Puente J,Valenzuela MA, Gamonal J. Levels of interleukin-17in gingival crevicular fluid and in supernatants of cel-lular cultures of gingival tissue from patients withchronic periodontitis. J Clin Periodontol 2005; 32:383-389.

12. Petrie A, Sabin C. Medical Statistics at a Glance.Blackwell Science 2000: 34-46, 68-69.

OHDMBSC - Vol. IV - No. 4 - December, 2005

25

Correspondence to: Dr. Cristina Gabriela Puºcaºu, Assistant Professor, Periodontology Disci-pline, Faculty of Dental Medicine and Pharmacy Constanþa. Home address: Siretului str., no. 54,900675, Constanþa, Romania. E-mail: cristinap@gmb.ro