J Clin Periodontol 1997: 24: 718-726 Phnfed in Denmark - AU rights reserved

Copyright Munksgaard 1997

ciiflical periodontology
n 0303-6979

The optimization of the BANA test as a screening instrument for gingivitis among subjects seeking dental treatment
Loesehe WJ, Kazor CE, Taylor GW: The optimization of the BAN,4 test as a screening instrument for gingivitis among subjects seeking dental treatment. J Clin Periodontol 1997; 24: 718-726, Munksgaard, 1997, Abstract. Porphyromonas gingivalis, Treponema denticola and Baeteroides forsythus have been implicated in periodontal disease and each possesses an enzyme capable of hydrolyzing the synthetic trypsin substrate, BANA. We have used a chairside test for BANA hydrolysis to diagnose an anaerobic periodontal infection in patients with advanced forms of clinical disease using a 15-min.'55C incubation protocol. However, the BANA test performance is dependent upon the length and temperature of incubation. In the present study, we have evaluated a 5-min/35C, a 5-mia'55C and a 15-min'55C incubation protocol to determine whether the performance of the BANA test could be optimized using plaque samples obtained from subjects seeking dental treatment. Logistic regression models were tested with age, smoking status, and gingivitis scores as covariates. The best fitting model obtained with the 5-min/35C protocol had a sensitivity of 71%, a specificity of 68%, a false-positive proportion of 9"/.), a false-negative proportion of 65%, and an overall accuracy of S(f/o. When maximum likelihood estimates were obtained in this model, plaques from individuals who reported that they currently smoked were 9.57 x , and those who quit smoking were 4.73 X more likely to have a positive BANA score than someone who never smoked. Plaques were 4.55x more likely to be BANA-positive if they were removed from sites with gingivitis. These findings indicate that the performance of the BANA test is best using the 5-min/35C incubation protocol.

Walter J, Loesche, Christopher E, Kazor and George W.Taylor

University ot Miciiigan School of Dentistry, Michigan, USA

Key words: BANA test; gingivitis; smokiiig Accepted for pubiicatjon 8 January 1997

Porphyromonas gingivalis, Treponema denticola and Baeteroides forsythus have been implicated in periodontal disease by cultural (Loesche et al, 1985, Loesche et al, 1992a, Dzink et al. 1988), by immunological (Loesche et al. 1992a, Simonson et al. 1992, Grossi et al, 1995) and by DNA probe (Loesche et al, 1992a, Haffajee et al, 1996, Lotufo et al, 1994) studies. These species are gram-negative anaerobes which possess, in vivo, an enzyme capable of hydroiyzing the synthetic trypsin substrate, Nbenzoyl-DL-arginine-2-naphthylamide (BANA). We have screened over 60 species of plaque bacteria for the presence of the BANA enzyme, and only these 3

species were uniformly strongly BANApositive, whereas several Baeteroides and Capnocytophaga species were occasionally BANA-positive, and then only when large numbers of CFUs were used (Loesche et al, 1990a). These considerations indicate that the detection of the BANA enzyme in plaque samples most likely reflects the presence of P. gingivalis, T. denticola and/or B. forsythus in these plaque samples. While the BANA test does not distinguish which of the three species are present, it appears from culture (Loesche et al. 1992a), DNA probe studies (Loesche et al. 1992a, Haffajee et al, 1996), and PCR studies (Ashimo-

to et al. 1996) that these species co-exist in high numbers in plaques obtained from deep pockets in periodontal patients. In these patients, from 90/) to 100% of the plaques removed from diseased sites are BANA-positive (Loesche et al. 1992a, Loesche et al. 1996). We have used this information to diagnose an anaerobic infection and have treated these patients with systemic metronidazole after completion of scaling and root planing (Loesche et al. 1996, Loesche & Giordano 1994), We have not used the BANA test as a screening instrument for periodontai disease because initial studies, using a 15-min/55C incubation protocol,

BANA test in gingivitis showed that many shallow sites in periodontal patients are often BANA-positive. However, Beck ct al. (1990) found in a random sample of older individuals, that a positive BANA test was both highly associated with the severity of attachment loss, and was a significant predictor of attachment loss over a 3-year period. This suggested that the BANA test might have some utility as a survey instrument for the presence of the BANA-positive bacteriai species in periodontal disease, if the number of false-positive results could be reduced. The BANA test, when incubated at 55C for 15 min, will detect about 10'' CFUs of these organisms (Loesche et al. 1992b). Thus it is possible that the test was configured to a level of analytic sensitivity in which it was detecting levels of these organisms which are not thought to be contributmg to clinical disease (Haffajee & Socransky 1994). These so-called false-positive reactions are commonly found with other techniques that have a high analytical sensitivity, such as DNA probes and immunological reagents (Loesche et al. 1992b, Savitt et ai, 1988, Lotufo et al. 1994, Wolfe 1992. Zambon et al, 1985), One way to decrease the false-positives and thereby improve the specificity of a test, would be to decrease the analytical sensitivity of the test. This can be easily done with the BANA test by decreasing either the length of incubation, or the temperature of incubation, or both. When the BANA test was evaluated at different times and temperatures, taking standard size plaque samples from all available interproximal sites in periodontally healthy individuals, falsepositive proportion of 34% was obtained when the plaque samples were incubated at 35C for 5-min compared to a false-positive proportion of 59% when the samples were incubated for 15-min. This suggested that incubating the BANA test for 5 min at 35C might improve its performance (Feitosa et al, 1993), In the present study, we have evaluated 3 time/temperature protocols for the BANA test, taking standardized plaque samples from the mesial interproximal surface of first molars that were present in the mouths of subjects appearing at a dental clinic seeking treatment. We used the level of gingivitis at the sample site, plus known risk factors for periodontal disease such as age and smoking (Bergstrom 1989. Grossi et al, 1995. Haber et al, 1993), lo model the BANA test scores in the three time/temperature protocols. Materials and Methods Consecutive subjects who appeared at the Patient Admitting and Emergency Services Clinic at the University of Michigan School of Dentistry were invited to participate in this study. After signing an informed consent, these patients were asked to complete a short questionnaire that surveyed age (date of birth), gender and smoking status, i.e., if the subject was a current smoker, a previous smoker or had never smoked. Previous smokers were asked the number of years since cessation, and current smokers were partitioned based on the number of cigarettes that they smoked per day, i,e,. &20 cigarettes. Cigarette. pipe and cigar smokers were combined in this study. Since periodontal disease most frequently begins subgingivally at the

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interdental papillae, periodontal health was determined at interproximal sites using the papillary bleeding score (PBS) (Loesche 1979), The PBS has been shown to be the most reliable of the several gingival bleeding indices that have been described (Marks et al, 1993), Proximal plaque was sampled from between the first molar and second premolar in each quadrant using a wooden toothpick (Stim-u-dent*. Johnson and Johnson Windsor, NJ), A separate wedge was used for each sampled site. If one site was absent, an adjacent interproximal area in the same quadrant was sampled. 2 lo 4 sampies from each of 508 patients were collected, yielding a total of 1976 plaques. The PBS has a 6-3evel scale with a PBS=O reflecting health, a PBS=1 indicating some degree of inflammation in the absence of bleeding and PBS scores of 2-5 reflecting different patterns of bleeding. Because the statistical tests used required that the conditions under

Table I Effect of Random assignment of subjects to BANA protocol on the frequency distribution o^ papillary bleeding scores (PBS) B,ANA time-temperature protocol PBS 0 1
T

5' 35C 76" 410 125 20 ^ 667 76 591 486 181 611 56 (lr:.;.)'(61%) (19%) (3%.) (5%) (99%) (11%) (89%) (73%) (27%) (92%) (8';-.,)

5' 55C 76 (12';i) 396 (62%) 105 (16%) 24 (4%) (6%) ^1 642 (!D0%) 76 566 472 170 577 65 (12%) (88'M,) (74'v;.) (26/o) (90%) (16%)

15 5 5 X {6"'..) (66%) (19/.) (3%) ^2 (6%) 667 (100%) 38 629 475 192 602 65 (6%) (94%) (71%) (29"40 (90%) (10%) 38 437 127 23

Total 190 1243 357 67 1976 190 1786 1433 543 (10/.l (90/n) (73%) (27%) (107c,) (63%) (18%) (3%) (6%)

3 4 0 i^ O-I 2-4 0-2 ,3-4 " No, sites. ''% sites.

1290 (91%) 186 (9%)

Table 2. Effect of BANA protocol on the frequency distribution of BANA reactions BANA reaction negative weak positive positive BANA-1 121 (18%) weak pos + pos BANA-2 neg-^weak pos positive No. sites, 5' 35C BANA time-temperature protocol ^' 55C 15' 55C 77 113 452 77 565 190 452 (12%) (18%) (70-<.) (12%) (88%) (30-.,) (70%) 79 (12%) 143 (21%) 445 (67%) 79 588 222 445 (12%) (88%) (33%) (67%)

Total 277 429 1270 277 1699 706 1270 (14%) (22%) (64"'.) (14%) (86%) (36'>.,) (64%)

(18%)'' 173 (26%) 373 (56%) 121 546 294 373 (18%) (82%,) (44';-..) (56%)

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Loesche et al. due to the presence of any one of the BANA-hydrolyzing bacteria diffused into the upper reagent strip where it reacted with the Evan's black dye to form a permanent blue-black color. Scores were assigned based on the amount of blue color visible on the upper reagent strip after each incubation protocol. A score of 1 (negative) was indicated when no blue color was visible, a score of 2 (weak-positive) when a faint blue color was noted, and a score of 3 (positive) when distinct blue color was observed. For analytic purposes, scores of 2 and 3 (BANA-1) were scored as positive in one analysis, and a score of 3 (BANA2) was scored as positive in another analysis, so as to determine which configuration was the best indicator of a positive response. In previous studies (Loesche et al. 1990a, Feitosa et a!. 1993, Amalfitano et al. 1993, Bretz et al. 1993. 'Watson et al. 1994), the BANA-1 configuration had been used, and it was possible that by including the weak-positive reactions with the positive scores, we were increasing the number of false-positive findings. The statistical modeling sought to determine which of the 3 time/temperature settings and 2 BANA score configurations gave optimal BANA test results after taking into account smoking status, PBS, and age. 6 logistic re-

Table 3. Distribution of smokers among the three BANA incubation protocols BANA time-temperature protocol Smoking History never quit current never ever never + quit current 5' 35C (n=667 sites) 46 32 46 54 68 32 5' 55C (n = 642 sites) 38 27 35 38 62 65 35 15' 55C (n=667 sites) 43 23 34 43 57 66 34 Total (n = 1976) 42 24 34 42 58 66 34

evaluation be dichotomous. it was necessary to recode the PBS for many of these analyses. Accordingly. 3 comparisons of gingival health were made PBS=O versus PBS= 1-4, PBS=O-1 versus P B S = 2 ^ . and PBS=0-2 versus PBS=3-4. The same investigator (CK) collected all plaque samples and judged each PBS. Adherent plaque present on the toothpick was analyzed for the presence of P. gingivalis, T. denticola and/or B. forsythus by using the BANA test. Each side of the wooden toothpick, after it was used to obtain the PBS in a single papillae, was wiped in a discrete location on the BANA impregtiated strip along the lower border of the BANA test card (Oral-B Laboratories, now

available from Knowell Periodontal Technologies. Toronto, Canada) (Feitosa et al. 1993). This method of sampling reduced the amount of sample size variability that is inherent when a curette is used to remove plaque. An upper reagent strip containing Evan's black dye was then activated through dampening with distilled water, and the two strips were folded over so they contacted one another. After folding, the card was inserted into one of three heating blocks and incubated at the designated protocol. The patient's BANA card was randomly assigned to one of three time/ temperature incubation groups; 5 min at 35C , 5 min at 55C or 15 min at 55C. Naphthylamide released

Tabie 4. Comparison of interactions between gingivitis fPBS), smoking status and BANA incubation temperature on BANA reactions BANA temperature setting U35C)I'' Gingivitis PBS comparison Positive BANA reactions Smoking status current versus (quit+ne^'er) never versus ever Current versus quit versus never Current versus (quit + never) never versus ever current versus uit versus never current versus (quit+never) never versus ever current versus quit versu.^ never current versus (quit+never) never versus ever current versus quit versus never current versus (quit + never) never versus ever current versus quit versus never current versus (quit+never) never versus ever current versus quit versus never BANA-1 Weak Pos+pos 88.3= BANA-2 positive :>" > > > > > S < < > > > > > > > > > 70.2' 106.7 107.1 52.4 92.1 93.0 524 97.0 97.6 26.8 30.7 34.0 48.2 51.7 56.2 37.3 40.5 45.3 differences S

10 versus ]^)l
0-1 versus 2-4

mrsi ! 114.01
72.7 97.4 101.5 61.0 93.4 96.3 41.1 50.4 54.0 61.1 70.3 75.5 42.! 50.3 55.7

0-2 versus 3-4

NS NS S

s s s s s

55C

0 versus 1-4

0-1 versus 2-4

(t-2 versus 3 - i

s s s s s s s s

Maximum likelihood ratio: al! values are significant a t / i < 0.0001. ^ Indicates which of the BANA score configurations was higher. S: significant; NS; not significant when the maximum likelihood statistics between the BANA-1 and the BANA-2 configurations were compared. * Values in box indicate the best value in column.

BANA test in gingivitis gression models were evaluated for each of the BANA test conditions. The age of the patient as a continuous variable and the smoking status of each patient, categorized as never smoked, ever smoked, (quit smoking pius current smoker), current smoker and quit smoking were included in the various models. The PBS was recoded into 3 groupings for comparative purposes, and each of these comparisons was entered into the model along with age and smoking status. All procedures were performed using the SAS* statistical programs (SAS Institute Inc.. Cary, NC). From the 54 models that were generated, the odds ratios, sensitivity, specificity, false-positive and faise-negative proportions were obtained for those models with the best fit (using -2 log likelihood statistic as the criterion). The final models were retested using generalized estimating equations (GEE) to determine the impact of observing correlated sites on the standard error estimates (Zeger & Liang 1986), 4 with all other PBS scores. Table 1 shows that the distributions of recoding of the PBS into two levels were comparable in each of the BANA protocols. The frequency of BANA-negative, weak-positive, or positive scores was evenly distributed among the three BANA incubation protocols (Table 2), The weak-positive score indicates lower numbers of the BANA-positive species, and for purposes of bivariate statistical analysis, it was combined with the BANA-positive scores in the original

721

evaluation of the BANA test. It is possible however that these weak-positive scores should be considered with the negative scores when the BANA score is referred to clinical status. In Table 2, the BANA-l designation was given for the standard way in which the BANA test was compared in prior studies (negative versus weak-positive + positive) and BANA-2 refers to the classification when the negative plus weak-positive scores are compared to the positive scores.

Table 5. Logistic regression model showing effect of gingivitis, smoking status and age on BANA reaction (negative versus weak-positive+positive) at 5' 35C Analysis of maximum likelihood estimates parameter estimate 0.3420 0.3420 1.9129 1.9129 L5094 1.5094 -0.021! -0.0211 standard error 0.3229 0.512 0.2588 0.354 0.2925 0.436 0.0065 0.010 95% confidence Interval

Variable Intercept Log. regression GEE eversmoke log. regression GEE PBS (0 versus 1-4) log. regression GEE Age log. regression GEE

p value 0.29

odds ratio

0.0001 <0.0001 0.0001 0.0005 0.0011 0.03

6.77 6.77 4.52 4.52 0.979 0.979

(4.08-11,25) (3.38-13.55) (2.55-8.03) (1.92-10.63) (0.97-0.99) (0.96-0.998)

Results
The 508 subjects included in this study ranged in age from 13 to 88 years with 51% being female. The random assignment of subjects to the 3 time/temperature incubation settings resulted in an equal distribution of subjects in terms of the variables under investigation, i.e., age, PBS (Table I) and smoking status (Table 3). The age of the subjects in the 5-min/35C group was 41.4 17.8 years; in the 5-min/55C group 40.5r7 years, and in the l5-min/55C group 41,9 yearsI7,4 years, 73% of the 1976 plaque samples were taken from interproximal sites that did not bleed when the toothpick wedge was inserted interproximally, i.e., a PBS of 0 or 1 (Table 1). Only 9% of the sampled sites reacted with a blood flow when the toothpick was inserted, i.e., a PBS of 3 or 4, The statistical procedures required that the PBS be recoded to 2 or 3 levels, 63% of the sites had a PBS of 1, meaning that, while the tissue was fibrotic or edematous, it did not bleed. Thus, in one of the contrasts we compared healthy sites with all diseased sites (PBS=O versus PBS=1 to 4), In another contrast, we compared the nonbleeding scores with the bleeding scores (PBS=O-1 versus PBS=2-4), Finally, we compared the sites in which the gingival bleeding, had a flow, i.e., PBS = 3 -

- 2 log likelihood=115.0 with 3 DF;i=0.0001. ROC area=0.799.

Table 6. Logistic regression rrtodei showing effect of gingivitis, smoking status (current versus quit versus never) and age on BANA reaction (negative versus weak-positive+positive) at 5' 35C ^ .^nalvsis of maximum likelihood estimates parameter estimate 0.2508 0.2508 2.2582 2.2582 1.5535 1.5535 1.5143 1.5143 -0.019 -0,019 standard error 0.3294 0.50! 0.3672 0.486 0.3318 0.431 0.2939 0.437 0.0066 0.009 0.0001 9.57 9.57 4.73 4.73 4.55 4.55 0.98 0.98 (4.66-19.65) (3.69-24.80) (2.47-9,06) (2.03-n.O) (2.56-8.09) (1.93-10.71) (0,97-0.994) (0.96-0.999) 95% confidence interval

Variable intercept log. regression GEE current smoker log. regression GEE quit smoking log. regression GEE PBS (0 versus 1^) log. regression GEE Age log. regression GEE - 2 Iog!ikelihood = ROC curve=0.798.

p-valuc

odd5 ratio

<o.ooai
0.0001 0.0003 0.0001 0,0005 0.0039 0.0404

with 4DFp=O.0O0l.

722

Loesche et al. 35C when the PBS=O was compared with the PBS of 1 to 4, for either when the "never smoked" group was compared with the "ever smoked" group, or when the "current smokers" were compared to the "quit smoking" and the "never smoked" groups. These 2 best candidate models, which also included age as a continuous variable in the analysis, were examined in more detail. Sites in individuals who reported that they currently smoked or who had quit smoking, i.e., the ever smoked group, were 6,11 x more likely to have a positive BANA score than sites in people who never smoked (Table 5), Sites with a PBS of ! to 4 were 4.52 X more likely to have a positive BANA score. Sites in older individuals were slightly less likely to have a positive BANA score. All odds ratios were significant, as the 95%. confidence interval did not include 1.0 (Table 5). The area under the receiver operator characteristic (ROC) curve was 0.799 indicating that the overall accuracy of the model was approximately SO*''.]. When the logistic regression tnodel was used with current smokers separated from those who had quit smoking, an equivalent likelihood ratio statistic and ROC curve area were obtained (Table 6). However, those individuals who were currently smoking were 9.57X as likely to have a BANA-positive score, whereas those who had quit smoking were 4.73 x more likely to have a BANA-positive score when both were compared to individuals who never smoked. Sites from individuals with some form of gingivitis were 4.55 X

Table 7, Frequency distribution of BANA test scores as a function of smoking status and PBS BANA test (BANA-n negative Gingivitis PBS=O PBS=l-4 total n = never smoked 31"., 69':-. 99 negative Gingivitis PBS=O PBS 1 ^ total n= never 31% 99 quit 15% 13 ever smoked
23"'o

Positive never smoked 12% 88% 207 positive ever smoked 4"-;.

2"."
339

cunent
33%

never 12% 207

quit 2% 28% 133

current 6% 24% 206

62%
9

The frequency distribution of BANA-1 and BANA-2 scores among the two 55C incubation protocols was comparable, i.e., about 88% of the plaques incubated for 5 or for 15 min were positive in the BANA-1 configuration and about 70% were positive in the BANA-2 configuration. Fewer plaques were BANA-positive at 35, as would be expected with the lower temperature, i.e., 82% in the BANA-1 configuration and 56% in the BANA-2 configuration (Table 2). The frequency distributions of PBS and BANA scores in Tables 1 and 2 were for interproximal sites not patients. In order to determine what these scores were on a per patient basis, we defined gingivitis as having either a cumulative PBS of ^ 4 for the 4 test sites, or having at least 1 site that bled, i.e., PBS &2. By these definitions about 85% of the subjects had a cumulative PBS s=4, and about 50 to 55% had one site which bleed. The prevalence of gingivitis as assessed by either method was equally distributed among the three BANA time/temperature protocols. We defined BANA colonization when 3 of the 4 sites were either weak-positive or positive, i.e., the BANA-1 configuration. About 84% of the subjects were classified as colonized with the BANApositive species when the plaque samples were incubated at 55C, and 76% were classified as colonized when the samples were incubated at 35C. 42% of the subjects reported never having smoked, while 24% had quit smoking and 34%. were current smokers. These smoking categories were evenly distributed among the 3 BANA incubation protocols (Table 3). When these smoking categories were recoded to "never smoked versus ever smoked" and to "current smokers ver-

sus never+quit" the frequency distribution of these groups was comparable in the 3 BANA incubation protocols. A series of multivariate logistic regression models were perfortned in which the time/temperature, the PBS, and the smoking status categories were varied, so as to determine their effect on the BANA scores using either the BANA-1 or the BANA-2 configuration. The modeling results for BANA scores, using the 15 min/55C incubation protocol, were the least significant statistically and will not be described further. All combinations of PBS scores and smoking status categories in either the 5-min/35C or 5-min/55C incubation protocol, were overwhelmingly related to the BANA scores ( / J < 0 . 0 0 0 1 ) (Table 4). The log likelihood ratio statistics were uniformly higher for the 5-min/ 35C protocol and for the BANA-1 configuration. The 2 highest log likelihood ratio statistics were obtained at

0.0

0,1

0,Z

0.4

0,5

O.G

0.7

0.8

0,3

1,0

I - Speciriclty
Fig. 1. Receiver operating characteristic curve for BANA test. A ROC plot of sensitivity versus [-specificity. The arrow indicates the cutpoint p value of 0.78 from which the model gives a sensitivity of 71.2% and a specificity of 67.8%.

BANA test in gingivitis more likely to have a positive BANA score, and older individuals were slightly less likely to have a positive BANA score. Again, the overall accuracy of the model as judged by area under the ROC curve was approximately 80%, These models were then evaluated using GEE. The random effects caused by the correlations among sites is reflected in the increased standard errors and slightly wider 95'^ confidence intervals (Tables 5, 6). However, these effects were not sufficiently different to yield conclusions that differed from those obtained using the ordinary logistic regression procedure. The frequency distribution of plaque BANA reactions in sites taken from health (PBS=O) and gingivitis (PBS= 14) as a function of smoking status, is shown in Table 7 for the models presented in Tables 5, 6. Almost all plaques from the gingivitis sites in smokers are BANA-positive, in agreement with the Odds Ratios shown in Tables 5 and 6. The log likelihood ratio statistics are comparable in the two models, i.e., 115 and 117,4, but because the "ever smoked" model is the more parsimonious of the two, with three degrees of freedom, we have chosen to display the sensitivity and specificity values for that model (Fig. 1, Table 7), The model has a sensitivity of 71.2%, a specificity of 67,8%. a false-positive proportion of 9,4%, and false-negative proportion of 65.1% when we set the cutpoint for the estimate of the probability of having a positive BANA test at 0.78 (the optimal reflection point on the ROC curve shown in Fig. 1). che & Giordano, 1994, Loesche et al. 1996). We have also found that the presence of BANA-positive plaques after completion of the hygienic phase of treatment, compared to the presence of BANA-negative plaques, can be significantly associated with the amount of attachment loss in the next 1 to 2 years (Loesche et al. 1990c). A positive BANA test immediately after the unsupervised usage of metronidazole can be associated with patient noncompliance (Loesche et al. 1993). These are useful considerations in the management of patients with periodontitis. In plaques from these patients it provides information on the presence of T. denlicota. B. forsytkus and P gingivalis that is comparable to that obtained by DNA probes and immunological reagents, and superior to that which can be obtained by culturing (Loesche et al. !992b|. However, none of these considerations implies that the BANA test could be useful as a screening instrument for these species in a group of individuals seeking dental care, let alone in the general population. In fact, because P gingivalis, T. denticola and B. forsythus can often be present in the absence of clinical disease, most procedures which can detect these species in low numbers such as DNA probes (Savitt et al. 1988, Lotufo et al. 1994), PCR assays (Ashimoto et al, 1996). immunological reagents (Loesche et al. )992a. Wolfe 1992, Zambon et al. 1985), and the BANA test (Loesche et al. 1990b), will give rise to an unacceptably high number of false-positive reactions when related to the clinical status of the sample site. The number of false-positives reflects that the BANA test used in the original 15 minutes/55C incubation protocol was detecting levels of P. gingivalis. B. forsythus and/or T, denticola, i.e., about lO-* CFUs (Loesche et al. 1992b), that are below the threshold for levels of these organisms that are associated with clinical disease (Haffajee & Socransky 1994). This detection level can be raised to about 10' CFUs by shortening the length of incubation and/or the temperature of incubation. Against this background, we evaluated the easily modifiable aspects of the BANA test to determine whether we could find a time/temperature setting that might optimize its performance as applied to gingivitis. When the BANA test was classified according to the BANA-l configur-

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Discussion
The BANA test was developed to substitute for the cuhuring and/or microscopic examination of plaque samples in order to identify certain periodontopathic species in plaques taken from patients with advanced forms of periodontal disease (Loesche et al. 1990a, 1990b, 1990c). In this role, from 90 to 100% of subgingival plaque samples taken by curette from deep pockets in periodontal patients are BANA-positive, indicating the presence of 7". denticola. P. gingivalis and B, forsythus (Loesche et al, 1992b). We have used this information to diagnose an anaerobic infection which can be successfully treated when metronidazole is added to the treatment regimen (Loes-

ation, 18% of the plaque samples were BANA-negative at 35C compared to 12% BANA-negative at 55X (Table 2). This slight shift in proportions of BANA-negative plaques resulted in considerably higher log likelihood ratio statistics for all BANA test incubated at 35C compared to 55C (Table 4), A similar magnitude of difference in log likelihood ratio statistic was also observed when the BANA reactions were classified according to the BANA-2 configuration. The magnitude of these differences indicated that the model clearly favored the 35C temperature. The model preferred the BANA-l configuration to the BANA-2 configuration, in that the log likelihood ratio statistic was higher for the BANAi configuration in 16 of the 18 comparisons with the BANA-2 configuration shown in Table 4, This suggested that the weak-positive BANA reactions are more akin to positive BANA reactions than to BANA-negative reactions. This means that levels of the BANA-positive species at the detection level of the BANA test, i,e., 10" to lO^ CFUs m a plaque sample, which we previously considered to reflect colonization, may be about the threshold of these organisms that can be associated with tissue inflammation. The models showed that in terms of obtaining the best fitting model, a PBS= I value, which accounted for 61% of all the PBS (Table 1), should be included among the diseased values (Table 4). We did not do this in previous studies as we combined the PBS = O and PBS= 1 together to give a value for nonbleeding sites (Loesche et al. 1990b, Bretz et al. 1993, Feitosa et al. 1993). The PBS = ! is identical to the G l = l of the widely used gingival index system (Loe & Silness 1963). This suggests that at this earhest clinical sign of gingivitis that the BANA-positive species are in the interproximal plaque. When the plaque was removed from a gingivitis site it was 4,5 X more likely to give a BANA-positive reaction than if it was removed from a healthy site (Tables 5. 6). This may be of some clinica! relevance as previous microbiological studies have associated the BANA-positive species. B. forsythus. P. gingivaiis and T. denticola with deep pockets in periodontal patients (Loesche et al. 1992b, Grossi et al, 1995, Ashimoto et al, 1996), The present findings indicate

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Loesche et al. ing peptides (Loesche 1993). When the GCF subsequently decreases causing a relative tissue anoxia, the anaerobic members of the plaque flora would be likely to survive and thus be selected for. Because most of these organisms are gram-negative, there would be an enrichment for lipopolysaccharides in the plaque which could account for an increase in gingival inflammation, i.e., PBS ssl (Tables 5,6). These data indicate that a positive BANA reaction in interproximal plaque samples is influenced by the incubation protocol, the gingival health of the sampled site, the individual's smoking status, and age. When these variables were included in various logistic regression models, very high log likelihood ratio statistics ranging from 26.8 to 117 were observed attesting to the validity of the model. The best models, obtained when the plaques were incubated for 5 min at 35C, had an accuracy of about 80%, but the sensitivity of 71% and specificity of 68% indicated that additional information is needed. These models showed that when plaque samples were taken from interproximal sites in individuals presenting at a dental school clinic seeking treatment, the BANA test was 9 times more likely to be positive in current smokers, and 4 x more likely to be positive in sites with gingivitis. This suggests that the BANA test, because of its ability to detect certain periodontopathic bacterial species in plaque samples, along with smoking status, age, and gingivitis might be of value in the periodontal risk assessment of individuals seeking dental treatment. In this regard, in a preliminary report, a BANA positive tooth site in adult subjects was four times more likely to subsequently loss attachment over a 12month period than was a BANA negative site (Paquette et al., as cited in Williams et. al., 1996). Zusammenfassung
Die Optimierung des BANA-Tesls als ein Screening Instrument fur Gingivitis hei Personen. die eine zalindrzllicite Behandlung suchen Porphyromonas gingivalis. Treponema denticola und Bacteroides forsythus wurden mit Parodonlalerkrankungen in Verbindung gebracht. und alle besitzen ein Enzym, das die Fahigkeit zur Hydrolyse des synthetisehen Substrates BANA hat. Bei Patienten mit ejner fortgeschrittenen Form der Parodontitis haben wir einen Chairside-Test zur BANAHydrolyse. Jedoch ist das Verhalten des BANA-Tests abhangig von der Lange und Temperatur der Inkubation. Um zu uberprufen, ob das Verhalten des BANA-Tests optimiert werden kann. haben wir in der vorliegenden Studie mit Plaqueproben von Patienten, die eine zahnarztliche Behandlung suchen, ein Inkubationsprotokoli von 5 Minuten.'35C. von i Minuten/55C und von 15 Minuten/55C bewertet. Logistische Regressionmodelle mit dem Alter, dem Rauchverhalten und den Gingivitiswerten als Kovarianten wurden getestet. Das am besten passende Model wurde mit dem 5-Minuten/ 35C-Modell erzielt. Es zeigte eine Sensitivitat von 71%, eine Spezifitat von 68%, einen Anteil an Falsch-Pcsitiven von 9%. einen .Antei! an Falsch-Negativen von 65% und eine Gesamtgenauigkeit von 80'%i. Wenn man in diesem Modell die maximalen Wahrseheinlichkeiten berechnet. so zeigte die Plaque von Personen. die berichteten. daB sie momentan rauchten. eine 9.57 fache und die von Personen. die mit dem Rauchen aufhorten die 4.73 fache Wahrscheinlichkeit filr einen positiven BANA-Test als die von jemandem. der nie rauchte. Plaque, die von einer Stelle entfernt wurde. die Gingivitis zeigte. hatte die 4.55 fache Wahr.scheinlichkeit fur einen positiven BANA-Test. Diese Ergebnisse zeigen, daB der BANA-Test am besten mit dem 5-Minuten.''35C-Modeil verlauft.

that the BANA-positive species have already appeared in the interproxima! plaque prior to the onset of bleeding. This confirms recent findings that indicates that the bacteria! flora in shallow pockets in periodontal patients is more like the flora of deep pockets in these patients than the flora found in shallow pockets in periodontally healthy subjects (Socransky . personal communication). This suggests that the detection of the BANA-positive species at the level of a non-bleeding gingivitis might be of value in terms of preventative strategies based upon hygiene procedures. Previous evaluations of the ability of the BANA test score to reflect gingivitis (Loesche et al. 1990b, Feitosa et al. 1993) did not take into account factors such as smoking which are now known to be risk factors for periodontal disease (Grossi et al. 1995, Bergstrom 1989, Haber et al. 1993). When smoking status was included in the regression models, those individuals who had "ever smoked" were over 6X more likely to have a positive BANA reaction than non-smokers (Table 5), and current smokers were over 9X more likely to have a BANA-positive reaction (Table 6). The relationship between smoking and the BANA-positive organisms probably reflects the biphasic effect of smoking on the gingival crevicular fluid (GCF) flow. Several investigators have reported reduced levels of GCF around the teeth in habitual smokers compared with nonsmokers (Hedin et al. 1981, Holmes 1990). However, during and immediately after smoking, there is an initial 2-fold increase of GCF followed within an hour to below baseline values (McLaughlin et al. 1993). This biphasic response reflects the effects of nicotine on the blood flow, in which initial peripheral vasodilatation is followed by vasoconstriction. These hemodynamic effects could perturb the subgingival plaque flora in ways that could both select for the BANA-positive species and cause inflammation. If the subgingival flora is host dependent for much of its nutrients, then the two-fold increase in GCF would cause a relative increase in bacterial numbers, especially those that can use host breakdown products from arginine-rich macromolecules such as collagen. In this environment, the BANA-positive species might have a competitive edge because they could immediately utilize most arginine contain-

Resume
Optimisation du test BANA comme instrument pour depisler la gingivite chez les sujets se presentant pour traitement dentaire Porphyromonas gingivalis. Treponema denticola et Bacteroides forsythus ont ete mis en cause dans la maladie parodontale et possedent chacun un enzyme capable d'hydrolyser le substrat de trypsine synthetique, BANA. Nous avons utilise un test de Thydrolyse de BANA a faire pres du fauteuiL pour diagnostiquer une infection parodontale anaerobie chez des patients presentant des formes avancees de la maladie clinique; le protocole utilise pour I'incubation etait de 15 min/55C. Cependant le fonctionnement du test BANA depend de la duree et de la temperature de I'incubation. Nous avons dans la presente etude evalue un protocole d'incubation de 5 min/35C, un autre de 5 min/55''C et un de 15 min/55C. pour determiner s'ii serait possible d'optimiser le fonctionnement du test

Acknowledgments This research was supported in part by a gift from Knowell Periodontal Technologies and NIDR Grant T35DE07101, Short-Term Training Grant, Health Professional Schools, Summer Research Fellowship. Carol Gerlach assisted in the preparation of the manuscript. Walter Loesche holds several patents on the BANA technology. This technology is licensed to Knowell Periodontai Technologies, Toronto, Canada.

BANA test in gingivitis

BANA: les echantillons de plaque utilises etaient preleves sur des sujets se presentani pour un traitemeni dentaire. Des modeles de regression logistique ont ete testes en utilisant Page. I'usage du tabac et les scores de la gingivite comme covariates. Le modele le mieux adapte. obtenu avec le protocole de 5 min/35X, avait une sensibilite de 71%, une specificite de 68%, une proportion de fauxpositift de 9%, une proportion de fau.ii-negatifs de 65%, et une precision d'ensemble de 80%. Dans ce modele, quand on obtenail des valeurs estimees pour la vraisemblance maximum, les echantillons de plaque de sujets disant fumer actuellement donnaient 9.57X et ceux qui arretaient de fumer 4.73 X plus vraisemblablement un score BAN.^'v positif qu"un sujet n'ayant jamais fume. Les plaques avaient 4.55 x plus vraisemblablement un BANA positif si elles provenaient de sites avec gingivite. Ces observations indiquent que le meilleur fonctionnement du test BANA s'obtient en utihsant un protocole d'incubatioD de 5 min/35C. bon, J. J. & Hausman, E (1995) Assessment of risk for periodontal disease (II). Risk indicators for alveolar bone loss. Journal of Periodontology 66, 23-29. Haber, J., Wattles. J., Crowley, M., Mandell, R.. Joshipura, K. & Kent, A. L. (1993) Evidence for cigarette smoking as a major risk factor for periodontitis. Journal of Periodontology M. 16-23. Haffajee, A. D. & Socransky, S. S. (1994) Microbial etiological agents of destructive periodontai diseases. Periodontology 2000 5.78-111 Haffajee, A. D, Dibart. S., Cugim, M. A., Smith, C Kent, R. L. Jr.. &Socransky, S. S.(1996) Biological effects of scaling and root planing. II Microbiological changes. Journal of Dental Research 75. (sp. issue, abstr. 940), 135. Hedin, C. A.. Ronquist. G.. & Forsberg. O. (1981) Cyciic nucleotide content in gingiva! tissue of smokers and non-smokers. Journal of Periodontal Research B16. 337343. Holmes. L. G. (1990) Effects of smoking and/or vitamin C on crevicular fluid flow in clinically healthy gingiva. Quintessence International 2\. 191-195. Loe, H. & Silness. J. (1963). Periodontal Disease in pregnancy. 1. Prevalence and severily. .4cta Odontohgia Scandinavia 21. 533551. Loesche, W J. (1979) Clinical and microbiological aspects of chemotherapeutic agents used according to the specific plaque hypothesis. Journal of Denial Research.. 58. 2404-2412. Loesche, W. J. (199,3) Bacterial mediators in periodontal disease. In Clinical Infectious Diseases Anaerobic bacteria and anaerobic infections. 16(suppl 4). S203-210. Loesche. W. J. & Giordano. J. R. (1994) Metronidazole in periodontitis v. Debridement should precede medication. Compendium Continuing Education in Dentistry 15. 1198-1217. Loesche, W J.. Bretz, W. A., Kerschensteiner, D.. Stoll, J. A.. Socransky. S. S.. Hujoel, P P & Lopatin. D. E. (1990a) The development of a diagnostic test for anaerobic periodontal infections based upon plaque hydrolysis of benzoyl-DL-arginine naphthylamide (BANA). Journal Clmical Microbiology 28. 1551-1559. Loesche, W J., Bretz, W. A.. Lopatin, D. E.. Stoll, J.. Rau. C. F . Hillenburg. K. C, Killoy. W. J.. Drisko. C. L., Wiiliams, R., Weber. H. P.. Clark. W. Magnusson, I., Wilker, C & Hujoel. P R < 1990b) Multicenter clinical evaluation of a chairside method for detecting certain periodontopathic bacteria in periodontal disease. Journal of Periodontologv 61, 189196. Loesche, W J., Giordano, J. & Hujoel, P P. (1990c) The utility of the BANA test for monitoring anaerobic infections due to spirochetes {Treponema denticola) in periodontal disease. Journal of Dental Research 69, 1696-1702.

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