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J Clin Periodontol 1 9 9 7 : 2 4 : 718-726Phnfed in Denmark - A U rights reserved Copyright Munksgaard 1

c i i f l i c a l p e r i o d o n t o l on 0303-6979

The op t im iza t i on o f t he BANAtes t as a sc reen ing i ns t rument f o rg ing i v i t i s among sub jec tsseek ing den ta l t rea tment

Walter J , Loesche,Christopher E, Kazor andGeorge W.TaylorU n i v e r s i t y ot M i c ii ig a n S c h o o l o f Dentistry,M i c h i g a n , U SA

Loesehe WJ, Kazor CE, Taylor GW: The optimization of the BA N,4 test as ascreening instrument for gingivitis among subjects seeking dental treatment.J C li n P e ri o d on to l 1 9 9 7 ; 2 4 : 7 1 8 - 7 2 6 , Munksgaard , 1997,A bstract. Porphyromonas gingivalis, Treponema denticola and Baeteroides for-sythus have been implicated in periodontal disease and each possesses an enzymecapable of hydrolyzing the synthetic trypsin substrate, BANA. We have used achairside test for BANA hydrolysis to diagnose an anaerobic periodontal infec-tion in patients with advanced forms of clinical disease using a 15-min.'55C in-cubation protocol. However, the BANA test performance is dependent upon thelength and temperature of incubation. In the present s tudy, we have evaluated a5-min/35C, a 5-mia'55C and a 15-min'55C incubation protocol to determinehether the performance of the BANA test could be optimized using plaqueamples obtained from subjects seeking dental treatment. Logistic regression

1 %, a specificity of 68% , a false-positiv e p ro po rti on of 9"/.), a false-negative pr o-65%, and an overall accuracy of S(f/o. When maximum likelihood

they currently sm oked were 9.57 x , an d those w ho quit sm oking were 4.73 Xues were 4 .55 x m ore likely to be BAN A-po sitive if they were removed from

K e y w o r d s : B A N A t e s t ; g i n g i v i ti s ; s m o k i i igA c c e p t e d f o r p u b i ic a t j o n 8 Ja n u a r y 1 99 7

and Baeteroides forsythus have

1994) studies. These species arean enzyme capable of hydroiyz-

species were uniformly strongly BANA-positive, whereas several Baeteroidesand Capnocytophaga species were oc-casionally BANA-positive, and thenonly when large numbers of CFUs wereused (Loesche et al, 1990a).These considerations indicate thatthe detection of the BANA enzyme inplaque samples most likely reflects thepresence of P. gingivalis, T. denticolaand /o r B. forsythus in these plaquesamples. While the BANA test does notdistinguish which of the three speciesare present, it appears from culture(Loesche et al . 1992a), DNA probe

to et al. 1996) that these species co-exiin high numbers in plaques obtainefrom deep pockets in periodontal ptients. In these patients, from 90/) 100% of the plaques removed from dieased sites are BANA-positive (Loeschet al. 1992a, Lo esche et al. 1996). Whave used this information to diagnoan anaerobic infection and have treatethese patients with systemic metronidzole after completion of scaling anroot planing (Loesche et al. 1996, Loeche & Giord ano 1994) ,We have not used the BA NA test

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BA NA test in g ingivitis 7showed that many shallow sites in peri-odontal patients are often BANA-posi-tive. However, Beck ct al. (1990) foundin a random sample of older individ-uals, that a positive BANA test wasboth highly associated with the severityof attachment loss, and was a signifi-cant predictor of attachment loss over a3-year period. This suggested that theBANA test might have some utility asa survey instrument for the presence ofthe BANA-positive bacteriai species inperiodontal disease, if the number offalse-positive results could be reduced.

The BANA test, when incubated at55C for 15 m in, will detect a bou t 10''CFUs of these organisms (Loesche etal . 1992b). Thus it is possible that thetest was configured to a level of analyticsensitivity in which it was detectinglevels of these organisms which are notthought to be contributmg to clinicaldisease (Haffajee & Socransky 1994).These so-called false-positive reactionsare commonly found with other tech-niques that have a high analytical sensi-tivity, such as DNA probes and im-munological reagents (Loesche et al.1992b, Savitt et ai, 1988, Lotufo et al.1994, Wolfe 1992. Za m bo n et al, 1985),One way to decrease the false-positivesand thereby improve the specificity of atest, would be to decrease the analyticalsensitivity of the test. This can be easilydone with the BANA test by decreasingeither the length of incubation, or thetemperature of incubation, or both.When the BANA test was evaluated atdifferent times and tem perat ures, tak-ing standard size plaque samples fromall available interproximal sites in peri-odontally healthy individuals, false-positive proportion of 34% was ob-tained when the plaque samples wereincubated at 35C for 5-min comparedto a false-positive proportion of 59%when the samples were incubated for15-min. This suggested that incubatingthe BA NA test for 5 min at 35C mightimprove its performance (Feitosa et al,1993),

In the present study, we have evalu-ated 3 time/tem perature p rotocols forthe BANA test, taking standardizedplaque samples from the mesial inter-proximal surface of first molars thatwere present in the mouths of subjectsappearing at a dental clinic seekingtreatment. We used the level of gingi-vitis at the sample site, plus known riskfactors for periodontal disease such asage and smoking (Bergstrom 1989.

lo model the BANA test scores in thethree time/temperature protocols .Materials and MethodsConsecutive subjects who appeared atthe Patient Admitting and EmergencyServices Clinic at the University ofMichigan School of Dentistry were in-vited to participate in this study. Aftersigning an informed consent, these pa-tients were asked to complete a shortquestionnaire that surveyed age (date ofbir th) , gender and smoking status, i.e.,if the subject was a current smoker, aprevious smoker or had never smoked.Previous smokers were asked the num-ber of years since cessation, and currentsmoke rs were partitio ned based on thenum ber of cigarettes that they smokedper day, i,e,. &20 cigarettes. Cigarette.pipe and cigar smokers were combinedin this study.

Since periodontal disease most fre-quently begins subgingivally at the

interdental papillae, periodontal heawas determined at interproximal susing the papillary bleeding score (P(Loesche 1979), The PBS has bshown to be the most reliable of the seral gingival bleeding indices that hbeen described (Marks et al, 199Proximal plaque was sampled from tween the first molar and second pmolar in each quadrant using a woodtoothpick (Stim-u-dent*. Johnson Johnson Windsor, NJ), A separwedge was used for each sampled sIf one site was absent, an adjacent inproximal area in the same quadrant wsampled. 2 lo 4 sampies from each508 patients were collected, yieldintotal of 1976 plaques.

The PBS has a 6-3evel scale witPBS=O reflecting health, a PBS=1 incating some degree of inflammationthe absence of bleeding and PBS scoof 2-5 reflecting different patterns bleeding. Because the statistical teused required that the conditions un

Table I Effect of Random assignment of subjects to BANA protocol on the frequency disbution o^ papillary bleeding scores (PBS)

B,ANA time-temperature protocolPBS01T34

5'76"

4 1012520

66 7

35C(lr:.;.)'-( 61 % )( 19% )

(3%.)( 5% )

( 99 % )

5'76

39610524^ 164 2

55C(12';i)( 62% )( 16% )

( 4 % )( 6% )( !D0% )

1538

4 3712723^ 2667

5 5 X{6"'..)

( 66%)(19/.)( 3% )( 6% )

( 100%)

Total190

124335767

1976

(107c,( 63 %( 18%

( 3%( 6%

0i ^O-I2 -40 -2,3-4

765914 86181611

56

( 11% )( 89% )( 73 % )( 27 % )( 92% )

(8';-.,)

765664 72170577

65

( 12% )(88'M,)(74'v;.)(26/o)( 90% )( 16 % )

3862 94 7519260 2

65

( 6% )(9 4 % )( 71% )(29"40( 90% )( 10 % )

1901786143354 3

1290186

(10/.(90/n( 73 %( 27%( 91 % )

( 9%" N o , sites.''% sites.

Table 2 . Effect of BANA protocol on the frequency distr ibution o f BANA react ionsB A N Areactionnegativeweak positivepositiveBANA-1121 ( 18 % )weak pos + posB A N A - 2neg-^weak p o spositive

5'

17337 312154 6

29437 3

B A N A35C(1 8 %) ' '( 26 % )( 56 % )

( 18% )(82% ,)

(44';-..)( 56% )

t ime- tempera ture^'

771134 52

7756 5

1904 52

55C( 12 % )( 18 % )(70-

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0 Loesche et al.3. Dist r ibu t ion of smokers among the th ree BANA incubat ion pro tocols

B A N A5 ' 35C

(n=667 sites)4 6324 6546832

t ime-temperature pro tocol5' 55C 15' 55C

(n= 642 sites) (n= 667 sites)38273538626535

4 323344 3576634

Total(n = 1976)4 224344 2586634

be dichotomous . it wasto recode the PBS for many

3 com-of gingival health were madeversus P B S = 1-4, P BS =O -1 ver-s P B S = 2 ^ . and P B S = 0 - 2 versus

The same investigator (CK)all plaque samples and judgedPBS.

Adherent plaque present on thewas analyzed for the presence

P. gingivalis, T. denticola and/or B.by using the BANA test. Each

of the wooden toothpick, after itto obtain the PBS in a singlewas wiped in a discrete loca-

on the BANA impregtiated s tr ipthe lower border of the BA N A

now

available from Knowell PeriodontalTechnologies. Toronto, Canada) (Feito-sa et al. 1993). This method of samplingreduced the amoun t of sample size vari-ability that is inherent when a curette isused to remove plaque. An upper re-agent strip containing Evan's black dyewas then activated through dam peningwith distilled water, and the two stripswere folded over so they contacted oneanother. After folding, the card was in-serted into one of three heating blocksand incubated at the designated pro-tocol.

The patient 's BANA card was ran-domly assigned to one of three time/temperature incubation groups; 5 minat 35C , 5 min at 55C or 15 min at55C. Naphthylamide released

due to the presence of any one of theBANA-hydrolyzing bacteria diffusedinto the upper reagent strip where it reacted with the Evan's black dye to forma permanent blue-black color. Scorewere assigned based on the amoun t ofblue color visible on the upper reagenstrip after each incubation protocol. Ascore of 1 (negative) was indicated whenno blue color was visible, a score of 2(weak-positive) when a faint blue colorwas noted, and a score of 3 (positive)when distinct blue color was observedFor analytic purposes, scores of 2 and3 (BANA-1) were scored as positive inone analysis, and a score of 3 (BANA-2) was scored as positive in anotheranalysis, so as to determine which con-figuration was the best indicator of apositive response. In previous studies(Loesche et al. 1990a, Feitosa et a!1993, Amalfitano et al. 1993, Bretz etal . 1993. 'Watson et al. 1994), theBANA-1 configuration had been usedan d it was possible that by including theweak-positive reactions with the posi-tive scores, we were increasing the num-ber of false-positive findings.The statistical modeling sought todetermine which of the 3 t ime/tempera-ture settings and 2 BANA score con-figurations gave optimal BANA test re-sults after taking into account smokingstatus, PBS, and age. 6 logistic re-

4. Co m p ar i so n of interactions between gingivitis fPBS), smoking status and BANA incubat ion temperature on BANA react ionsB A N AtemperaturesettingU35C)I''

55C

GingivitisPB Scomparison10 versus ]^)l

0-1 versus 2-4

0-2 versus 3-4

0 versus 1-4

0-1 versus 2-4

(t-2 versus 3-i

Smoking s tatuscurren t versus (quit+ne^'er)never versus everCurren t versus quit versus neverCurrent versus (quit + never)never versus evercurren t versus uit versus nevercurren t versus (qu i t+never)never versus evercurren t versus quit versu.^ nevercurren t versus (qu i t+never)never versus evercurren t versus quit versus nevercurren t versus (quit + never)never versus evercurren t versus quit versus nevercurren t versus (qu i t+never)never versus evercurren t versus quit versus never

Positive BANABANA-1

Weak Po s+p o s88.3= :mrsi! 114.0172.797.4

101.561.093.496.341.150.454.061.170.375.54 2 . !50.355.7

reactionsBANA-2positive

>" 70.2'> 106.7> 107.1> 52.4> 92.1> 93.0S 524< 97.0< 97.6> 26.8> 30.7> 34.0> 48.2> 51.7> 56.2> 37.3> 40.5> 45.3

differences

SsssssNSN S

SssssssssMaximum l ikel ihood rat io : al! values are significant at/ i< 0.0001.Indicates which of the BAN A score conf igurations was higher.S: significant; NS; not significant when the maximum likelihood statistics between the B A N A - 1 and the BA NA -2 configurations were

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B A N A t e s t in gingivit is 7gression models were evaluated for eachof the BANA test conditions. The ageof the patient as a continuous variableand the smoking status of each patient,categorized as never smoked, eversmoked, (quit smoking pius currentsmoker), current smoker and quitsmoking were included in the variousmodels . The PBS was recoded into 3groupings for comparat ive purposes ,and each of these comparisons wasentered into the model along with ageand smoking status. All procedureswere perform ed using the SAS* statis ti-cal programs (SAS Institute Inc..Cary, NC). From the 54 models thatwere generated, the odds ratios, sensi-tivity, specificity, false-positive andfaise-negative proportions were ob-tained for those models with the best fit(using -2 log likelihood statistic as thecriterion). The final models were re-tested using generalized estimatingequat ions (GEE) to determine the im-pact of observing correlated sites on thestandard error estimates (Zeger & Li-ang 1986),

ResultsT he 508 subjects included in this studyranged in age from 13 to 88 years with51% being female. The random assign-ment of subjects to the 3 t ime/ tempera-ture incubation settings resulted in anequal dis tr ibution of subjects in termsof the variables under investigation, i.e.,age, PBS (Table I) and smoking status(Table 3). The age of the subjects in the5-min/35C group was 41.4 17.8 years;in the 5-min/55C group 4 0. 5 r7 years ,and in the l5-min/55C group 41,9year s I7 , 4 year s,

73% of the 1976 plaque samples weretaken from interproximal sites that didnot bleed when the toothpick wedgewas inserted interproximally, i.e., a PBSof 0 or 1 (Table 1). Only 9% of thesampled sites reacted with a blood flowwhen the toothpick was inserted, i.e., aPBS of 3 or 4, The statistical proceduresrequired that the PBS be recoded to 2or 3 levels, 63% of the sites had a PBSof 1, meaning that, while the tissue wasfibrotic or edematous, it did not bleed.Thus , in one of the contras ts we com-pared healthy sites with all diseasedsites (PBS=O versus P B S = 1 to 4), Inanother contras t , we compared the non-bleeding scores with the bleeding scores(PBS=O-1 versus PBS =2-4 ) , F inal ly,

4 wi th all other PBS scores. Table 1shows that the distributions of recodingof the PBS into two levels were compar-able in each of the BANA protocols .The frequency of BANA-negative,weak-positive, or positive scores wasevenly distributed among the threeBANA incubation protocols (Table 2),The weak-positive score indicates lower

numbers of the BANA-positive species,and for purposes of bivariate statisticalanalysis , it was combined with theBANA-positive scores in the original

evaluation of the BANA test. It is poible however that these weak-positscores should be considered withnegative scores when the BANA scorreferred to clinical statu s. In Table 2, BANA-l designation was given for s tandard way in which the BANA twas compared in prior studies (negatversus weak-positive + po sitive) aBANA-2 refers to the classificatwhen the negative plus weak-positscores are compared to the positscores.

Table 5. Logistic regression model showing effect of gingivitis, smoking status and ageBANA reaction (negative versus weak-positive+positive) at 5' 35CAnalysis of maximum likelihood estimates

Variable parameterestimatestandarderror p value odds ratio

95%confidencIntervalInterceptLog. regression

G E Eeversmoke

log. regressionG E EPB S (0 versus 1-4)log. regressionG E EAgelog. regression

G E E

0.34200.34201.91291.9129

L50941.5094

- 0 . 0 2 1 !-0 . 0211

0.32290.5120.25880.3540.29250.4360.00650.010

0.29

0.0001

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72 2 Loesche et al.Table 7 , Frequency dis tribution of BANA test scores as a function of smoking s tatus andPBS

GingivitisPBS=OP B S = l - 4to ta l n =

GingivitisPBS=OP B S 1 ^total n=

negativeBANA test

never smoke d ever smoked31".,69':-.99

never31%99

negativequit15%13

23"'o

cunent33%6 2%9

(BANA-nPositive

never smoked ever smoked12%88%

207

never12 %

207

positivequit

2%28%

133

4"-;.2""339

current6%

24 %206

The frequency distr ibution ofBANA-1 and BANA-2 scores amongthe two 55C incubation protocols wascomp arable, i .e ., about 88% of theplaqu es incub ated for 5 or for 15 minwere positive in the BANA-1 configur-ation and abou t 70% were posit ive inthe BANA-2 configuration. Fewerplaques were BANA-positive at 35, aswould be expected with the lower tem-perature, i .e ., 82% in the BANA -1 con-f iguration and 56% in the BANA -2 con-figuration (Table 2).The frequency distributions of PBSand BA NA scores in Tables 1 an d 2were for interproximal sites not pa-tients. In order to determine what thesescores were on a per patient basis, wedefined gingivitis as having either acumulative PBS of ^ 4 for the 4 testsites, or hav ing at least 1 site th at b led,i.e., PBS &2. By these definitions about

85% of the subjects had a cumulativePBS s=4, and ab out 50 to 55% had onesite which bleed. The prevalence of gin-givitis as assessed by either method wasequally distr ibuted among the threeBANA time/temperature protocols. Wedefined BAN A colonization when 3 ofthe 4 sites were either weak-positive orpositive, i.e., the BANA-1 configur-ation. Ab ou t 84 % of the subjects wereclassified as colonized with the BANA-positive species when the plaquesamples were incubated at 55C, and76% were classified as colonized whenthe samples were incubated at 35C.

4 2% of the subjects reported neverhaving smoked, while 24 % had qui tsmok ing and 34% . were currentsmokers. These smoking categorieswere evenly distributed among the 3BANA incubation protocols (Table 3) .When these smoking categories were re-coded to "never smoked versus eversmoked" and to "cur rent smokers ver-

su s never+quit" the frequency distr i-bution of these groups was comparablein the 3 BAN A incubation protocols.A series of multivariate logistic re-gression models were perfortned inwhich the t ime/temperature, the PBS,and the smoking status categories werevaried, so as to determine their effecton the BANA scores using either theBANA-1 or the BANA-2 configuration.The modeling results for BANA scores,using the 15 min/55C inc ubation pro -tocol, were the least significant statisti-cally and will not be described further.All combinations of PBS scores andsmoking status categories in either the5-min/35C or 5-min/55C incubationprotocol, were overwhelmingly relatedto the BANA scores ( / J < 0 . 0 0 0 1 ) (Table4 ). The log likelihood ratio statisticswere uniformly higher for the 5-min/35C protocol and for the BANA-1configuration. The 2 highest log likeli-hood ratio statistics were obtained at

35C when the PBS=O was comparwith the PBS of 1 to 4, for either whthe "never smoked" group was compared with the "ever smoked" group, when the "current smokers" were compared to the "quit smoking" and t"never smoked" groups.These 2 best cand idate m odels, whialso included age as a continuous varable in the analysis, were examined more detail. Sites in individuals who rported that they currently smoked who had quit smoking, i.e., the evsmoked group, were 6 ,1 1 x more liketo have a positive BANA score thasites in people who never smoked (Tab

5), Sites with a PBS of ! to 4 we4.52 X more likely to have a positiBANA score. Sites in older individuawere slightly less likely to have a postive BANA score. All odds ratios wesignificant, as the 95%. confidence inteval did not include 1.0 (Table 5). Tarea under the receiver operator charateristic (ROC) curve was 0.799 indcating that the overall accuracy of tmodel was approximately SO*''.].

When the logistic regression tnodwas used with current smokers sepaated from those who had quit smokinan equivalent likelihood ratio statistand ROC curve area were obtaine(Table 6). However, those individuawho were currently smoking we9.57X as likely to have a BA NA -postive score, whereas those who had qusmo king w ere 4 .73 x more likely to haa BANA-posit ive score when both wecompared to individuals who nevsmoked. Sites from individuals wisome form of gingivitis were 4.55

0 . 4 0 , 5 O.G 0 . 7 0 . 8 0 , 3 1 ,0 . 0 0 , 1 0 , ZI - Speciriclty

Fig. 1 . Receiver operat ing chara cteristi c curve for BAN A test. A ROC plot of sensitivity ve rs[-specificity. The arrow indicates the cutpoint p value of 0.78 from which the model givessensitivity of 71.2% and a specificity of 67.8% .

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BA NA test in gingiv i tis 72 3more likely to have a positive BANAscore, and older individuals wereslightly less likely to have a positiveBANA score. Again, the overall accu-racy of the model as judg ed by areaunder the ROC curve was approximate-ly 80% ,

These models were then evaluatedusing GEE. The random effects causedby the correlations among sites is re-flected in the increased standard errorsand slightly wider 95 '^ confidence inter-vals (Tables 5, 6). However, these effectswere not sufficiently different to yieldconclusions that differed from those ob-tained using the ordinary logistic re-gression procedure.

The frequency distribution of plaqueBANA reactions in sites taken fromhealth (PBS=O) and gingivitis (PBS= 1 -4) as a function of smoking status, isshown in Table 7 for the models pre-sented in Tables 5, 6. Alm ost all pla quesfrom the gingivitis sites in smokers areBANA-positive, in agreement with theOdds Ratios shown in Tables 5 and 6.The log likelihood ratio statistics arecomparable in the two models, i.e., 115and 117,4, but because the "eversmoked" model is the more parsimoni-ous of the two, with three degrees offreedom, we have chosen to display thesensitivity and specificity values for thatmod el (Fig. 1, Table 7), Th e model hasa sensitivity of 71.2% , a specificity o f6 7 ,8 % . a false-positive proportion of9,4% , and false-negative p roportion of65.1% when we set the cutpoint for theestimate of the probability of having apositive BANA test at 0.78 (the optimalreflection point on the ROC curveshown in Fig. 1).

DiscussionThe BANA test was developed to sub-sti tute for the cuhuring and/or micro-scopic examination of plaque samplesin order to identify certain peri-odontopathic species in plaques takenfrom patients with advanced forms ofperiodontal disease (Loesche et al.1990a, 1990b, 1990c). In this role, from90 to 100% of subgingival plaquesamples taken by curette from deeppockets in periodontal patients areBANA-positive, indicating the presenceof 7". denticola. P. gingivalis an d B, for-sythus (Loesche et al, 1992b). We haveused this information to diagnose ananaerobic infection which can be suc-cessfully treated when metronidazole isadded to the treatment regimen (Loes-

che & Giordano, 1994, Loesche et al.1996). We have also found that the pres-ence of BANA-positive plaques aftercompletion of the hygienic phase oftreatment, compared to the presence ofBANA-negative plaques, can be sig-nificantly associated with the amount ofattach men t loss in the next 1 to 2 years(Loesche et al. 1990c). A positiveBANA test immediately after the un-supervised usage of metronidazole canbe associated with patient noncompli-ance (Loesche et al. 1993).

These are useful considerations in themanagement of patients with peri-odontitis. In plaques from these pa-tients it provides information on thepresence of T. denlicota. B. forsytkusan d P gingivalis that is comparable tothat obtained by DNA probes and im-munological reagents, and superior tothat which can be obtained by culturing(Loesche et al. !992b|. However, noneof these considerations implies that theBANA test could be useful as a screen-ing instrument for these species in agroup of individuals seeking dentalcare, let alone in the general popula-tion. In fact, because P gingivalis, T.denticola an d B. forsythus can often bepresent in the absence of clinical dis-ease, most procedures which can detectthese species in low numbers such asDNA probes (Savitt et al. 1988, Lotufoet al. 1994), PCR assays (Ashimoto etal , 1996). immunological reagents (Loe-sche et al. )992a. Wolfe 1992, Zambonet al. 1985), and the BANA test (Loes-che et al. 1990b), will give rise to an un-acceptably high number of false-posi-tive reactions when related to the clin-ical status of the sample site.

The number of false-positives reflectsthat the BANA test used in the original15 minutes/55C incub ation p rotoco lwas detecting levels of P. gingivalis. B.forsythus and/or T, denticola, i.e., aboutlO-* CFUs (Loesche et al. 1992b), thatare below the threshold for levels ofthese organisms that are associatedwith clinical disease (Haffajee & Soc-ransky 1994). This detection level canbe raised to about 10' CFUs by short-ening the length of incubation and/orthe temperature of incubation. Againstthis background, we evaluated the eas-ily modifiable aspects of the BANA testto determine whether we could find atime/temperature sett ing that mightoptimize its performance as applied togingivitis.

When the BANA test was classifiedaccording to the BANA-l configur-

ation, 18% of the plaque samples werBANA-negative at 35C compared t12% BANA-negative at 55 X (Tabl2) . This slight shift in proportions oBANA-negative plaques resulted inconsiderably higher log likelihoodratio statistics for all BANA test incubated at 35C compared to 55C(Table 4), A similar magnitude of difference in log likelihood ratio statistiwas also observed when the BANA reactions were classified according to thBANA-2 configuration. The magnitude of these differences indicated thathe model clearly favored the 35Ctemperature.

The model preferred the BANA-configuration to the BANA-2 configuration, in tha t the log likelihoodratio statistic was higher for the BANAi configuration in 16 of the 18 com parisons with the BANA-2 configurationshown in Table 4, This suggested thathe weak-positive BANA reactions armore akin to posit ive BANA reactionthan to BANA-negative reactions. Thimeans that levels of the BANA-positivspecies at the detection level of thBA NA test, i,e., 10" to lO^ C FU s m plaque sample, which we previouslconsidered to reflect colonization, mabe about the threshold of these organisms that can be associated with tissuinflammation.

The models showed that in terms oobtaining the best fitting model, PBS= I value, which accounted for 61%of all the PBS (Table 1), should be included among the diseased value(Table 4 ). We did no t d o this in previoustudies as we combined the PBS = O anP B S = 1 together to give a value for nobleeding sites (Loesche et al. 1990bBretz et al. 1993, Feitosa et al. 1993)T he PBS = ! is identical to the G l = l othe widely used gingival index system(Loe & Silness 1963). This suggests thaat this earhest clinical sign of gingivitithat the BANA-positive species are inthe interproximal plaque. When thplaque was removed from a gingivitisite it was 4,5 X more likely to give BANA-positive reaction than if it waremoved from a healthy site (Tables 56) .

This may be of some clinica! relevance as previous microbiologicastudies have associated the BANA-positive species. B. forsythus. P. gingivaiian d T. denticola with deep pockets inperiodontal patients (Loesche et al1992b, Grossi et al, 1995, Ashimoto eal, 1996), The present findings indicat

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72 4 Loesche et al.that the BANA-positive species have al-ready appeared in the interproxima!plaque prior to the onset of bleeding.This confirms recent findings that indi-cates that the bacteria! flora in shallowpockets in periodontal patients is morelike the flora of deep pockets in thesepatients than the flora found in shallowpockets in periodontally healthy sub-jects (Socransky . personal com mun i-cation). Th is suggests that the detectionof the BANA-positive species at thelevel of a non-bleeding gingivitis mightbe of value in terms of preventativestrategies based upon hygiene pro-cedures.

Previous evaluations of the ability ofthe BANA test score to reflect gingivitis(Loesche et al. 1990b, Feitosa et al.1993) did not take into account factorssuch as smoking which are now knownto be risk factors for periodontal dis-ease (Grossi et al. 1995, Bergstrom1989, Haber et al. 1993). When smok-ing status was included in the regressionmodels , those individuals who had"ever smoke d" were over 6X morelikely to have a positive BANA reactionthan non-smokers (Table 5), and cur-rent sm okers were over 9X m ore likelyto have a BANA-positive reaction(Table 6).

The relationship between smokingand the BANA-positive organismsprobably reflects the biphasic effect ofsmoking on the gingival crevicular fluid(GCF) flow. Several investigators havereported reduced levels of GCF aroundthe teeth in habitual smokers comparedwith nonsmokers (Hedin et al . 1981,Holmes 1990). However, during and im-mediately after smoking, there is an in-itial 2-fold increase of G C F followedwithin an hour to below baseline values(McLaughlin et al. 1993). This biphasicresponse reflects the effects of nicotineon the blood flow, in which initial pe-riphe ral vaso dilata tion is followed byvasocons tr ic t ion . These hemodynamiceffects could perturb the subgingivalplaque flora in ways that could both se-lect for the BANA-positive species andcause inflammation. If the subgingivalflora is host dependent for much of itsnutrients, then the two-fold increase inGCF would cause a relative increase inbacterial numbers, especially those thatcan use host breakdown products fromarginine-rich macromolecules such ascollagen. In this environment, theBANA-positive species might have apetitive edge because they could im-

ing peptides (Loesche 1993). When theGCF subsequently decreases causing arelative tissue anoxia, the anaerobicmembers of the plaque flora would belikely to survive and thus be selectedfor. Because most of these organismsare gram-negative, there would be anenrichment for l ipopolysaccharides inthe plaque which could account for anincrease in gingival inflammation, i.e.,PBS ssl (Tables 5,6).

These data indicate that a positiveBANA reaction in interproximal plaquesamples is influenced by the incubationprotocol, the gingival health of thesampled site, the individual's smokingstatus, and age. When these variableswere included in various logistic re-gression models, very high log likeli-hood ratio statistics ranging from 26.8to 117 were observed attesting to thevalidity of the model. The best models ,obtained when the plaques were incu-bated for 5 min at 35C, had an ac cu-racy of abo ut 80 % , but the sensitivity of71% and specificity of 68% indicatedthat additional information is needed.These models showed that when plaquesamples were taken from interproximalsites in individuals presenting at a den-tal school clinic seeking treatment, theBA NA test was 9 times m ore likely tobe positive in current smokers, and 4 xmore likely to be positive in sites withgingivitis. This suggests that the BANAtest, because of its ability to detect cer-tain periodontopathic bacterial speciesin plaque samples, along with smokingstatus, age, and gingivitis might be ofvalue in the periodontal r isk assessmentof individuals seeking dental treatment.In this regard, in a preliminary report,a BANA positive tooth s ite in adultsubjects was four times more likely tosubsequently loss attachment over a 12-mo nth period than was a BANA nega-tive site (Paquette et al., as cited in Wil-liams et. al., 1996).

AcknowledgmentsThis research was suppo rted in part by agift from Knowell Periodontal Techno-logies and NIDR Grant T35DE07101,Short-Term Training Grant, Health Pro-fessional Schools, Summer ResearchFellowship. Carol Gerlach assisted in thepreparation of the manuscript. WalterLoesche holds several patents on theBANA technology. This technology is li-

ZusammenfassungDie Optimierung des BA NA -Tesls als Screening Instrument fur Gingivitis hei Penen. die eine zalindrzllicite Behandlung sucPorphyromonas gingivalis. Treponema decola und Bacteroides forsythus wurden Parodonla lerkrankungen in Verbindung bracht. und alle besitzen ein Enzym, dasFahigkeit zur Hydrolyse des synthetiseSubstrates BANA hat. Bei Patienten mitner fortgeschrittenen Form der Parodonhaben wir einen Chairside-Test zur BAHydrolyse. Jedoch ist das Verhalten BANA-Tests abhangig von der Lange Temperatur der Inkubat ion. Um zu uberpfen, ob das Verhalten des BANA-Tests omiert werden kann. haben wir in der vorgenden Studie mit Plaqueproben von Patten, die eine zahnarztliche Behandlsuchen, ein Inkubationsprotokoli von 5 nuten.'35C. von i Minuten/55C und vonMinuten/55C bewertet. Logistische Regsionmodelle mit dem Alter, dem Rauchvhalten und den Gingivitiswerten als Kovanten wurden getestet. Das am besten psende Model wurde mit dem 5-Minu35C-Modell erzielt. Es zeigte eine Sensittat von 7 1% , eine Spezifitat von 68% , eiAnteil an Falsch-Pcsitiven von 9 % . ei.Antei! an F alsch-Negativen von 65% eine Gesa mtg enau igke it von 80'% i. Wenn min diesem Modell die maximalen Waseheinlichkeiten berechnet. so zeigte die Pque von Personen. die berichteten. daB momentan rauchten. eine 9.57 fache und von Personen. die mit dem Rauchen aufhten die 4.73 fache Wahrscheinlichkeit filrnen positiven BANA-Test als die von jemdem. der nie rauchte. Plaque, die von eiStelle entfernt wurde. die Gingivitis zeihatt e die 4 .55 fache Wahr.scheinlichkeit einen positiven BANA-Test. Diese Ergebse zeigen, daB der BA NA-Test am besten dem 5-Minuten.''35C-Modeil verlauft.

ResumeOptimisation du test BA NA comme insment pour depisler la gingivite chez les suse presentant pour traitement dentairePorphyromonas gingivalis. Treponema decola et Bacteroides forsythus ont ete mis cause dans la maladie parodontale et posdent chacun un enzyme capable d'hydrolyle substrat de trypsine synthetique, BANNous avons utilise un test de Thydrolyse BANA a faire pres du fauteuiL pour diagntiquer une infection parodontale anaerochez des patients presentant des formes avcees de la maladie clinique; le protocole utse pour I 'incubation etait de 15 min/55Cep endan t le fonctionnem ent du test BANdepend de la duree et de la temperature I 'incubation. Nous avons dans la preseetude evalue un protocole d'incubation demin/35C, un autre de 5 min/55''C et un de

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BANA test in gingivitis 7BANA: les echantillons de plaque utilisesetaient preleves sur des sujets se presentanipour un traitemeni dentaire. Des modeles deregression logistique ont ete testes en utili-sant Page. I'usage du tabac et les scores dela gingivite comme covariates. Le modele lemieux adapte. obtenu avec le protocole de 5min/35X, avait une sensibilite de 71%, unespecificite de 68%, une proportion de faux-positift de 9%, une proportion de fau.ii-nega-tifs de 65%, et une precision d'ensemble de80%. Dans ce modele, quand on obtenail desvaleurs estimees pour la vraisemblance maxi-mum, les echantillons de plaque de sujets di-sant fumer actuellement donnaient 9.57X etceux qui arretaient de fumer 4.73 X plus vrai-semblablement un score BAN. v positif qu"unsujet n'ayant jamais fume. Les plaquesavaient 4.55 x plus vraisemblablement unBANA positif si elles provenaient de sitesavec gingivite. Ces observations indiquentque le meilleur fonctionnement du testBANA s'obtient en utihsant un protocoled'incubatioD de 5 min/35C.

ReferencesAmalfitano. J.. de Fillippo, A. B.. Bretz. W

A. & Loesche. W }. (1993) The effects ofincubation length and temperature on thespecificity and sensitivity of the BANA (N-t>enzoyl-DL-arginine-naphthylamide) test.Journal of Periodontology 64, 848-852.

Ashimoto. A.. Chen. C. Bakker. I. & Slots,J. (1996) Polymerase chain reaction detec-tion of S putative periodontal pathogens insubgingival plaque of gingivitis and ad-vanced periodontitis lesions. Oral Micro-biology and Immunology 11, 266-273.

Beck. J. D., Koch, G. C, Rozier, R. G. &Tudor. G. E. (1990) Prevalence and risk in-dicators for periodontal attachment loss ina population of older community dwellingblacks and whites. Journal of Periodonto-logy 6\. 52l-52i.

Bergstrom, J. (19891 Cigarette smoking asrisk factor in chronic periodontai disease.Community Dentistry. Oral Epidemiology17, 245-247.

Bretz, W A., Ekiund. S. A.. Radicchi. R..Schork, A. & Loesche. W. J. (1993) Theuse of a rapid enzymatic assay in the fieldfor the detection of infections associatedwith adult periodontitis. Journal of PublicHealth Dentistry. 53. 235-240.

Dzink. J. L., Socransky. S. S. & Haffajee, A.D. (1988) The predominant cultivablemicrobiota of active and inactive lesions ofdestructive periodontal diseases. Journalof Clinical Periodontology 15. 316-323.

Feitosa, A. C. R.. Amalfitano. J. & Loesche.W. ]. (1993) The effect of incubation tem-perature on the specificity of the BANA(N-benzoyl-DL-arginine-naphthylamide)test. Oral Microbiology. Oral Immunology8, 57-61 .

bon, J. J. & Hausman, E (1995) Assess-ment 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 majorrisk factor for periodontitis. Journal ofPeriodontology M. 16-23.

Haffajee, A. D. & Socransky, S. S. (1994) Mi-crobial etiological agents of destructiveperiodontai diseases. Periodontology 20005.78-111

Haffajee, A. D, Dibart. S., Cugim, M. A.,Smith, C Kent, R. L. Jr.. &Socransky, S.S.(1996) Biological effects of scaling androot 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 gingi-va! tissue of smokers and non-smokers.Journal of Periodontal ResearchB16. 337-343 .

Holmes. L. G. (1990) Effects of smokingand/or vitamin C on crevicular fluid flowin clinically healthy gingiva. QuintessenceInternational 2\. 191-195.

Loe, H. & Silness. J. (1963). Periodontal Dis-ease in pregnancy. 1. Prevalence and sever-ily. .4cta Odontohgia Scandinavia 21 . 533-551.

Loesche, W J. (1979) Clinical and microbio-logical aspects of chemotherapeutic agentsused according to the specific plaque hypo-thesis. Journal of Denial Research.. 58.2404-2412.

Loesche, W. J. (199,3) Bacterial mediators inperiodontal disease. In Clinical InfectiousDiseases Anaerobic bacteria and anaer-obic infections. 16(suppl 4). S203-210.

Loesche. W. J. & Giordano. J. R. (1994) Me-tronidazole in periodontitis v. Debride-ment should precede medication. Compen-dium Continuing Education in Dentistry 15.1198-1217.

Loesche, W J.. Bretz, W. A., Kerschensteiner,D.. Stoll, J. A.. Socransky. S. S.. Hujoel, PP & Lopatin. D. E. (1990a) The develop-ment of a diagnostic test for anaerobicperiodontal infections based upon plaquehydrolysis of benzoyl-DL-arginine naph-thylamide (BANA). Journal ClmicalMicrobiology 28. 1551-1559.

Loesche, W J., Bretz, W. A.. Lopatin, D. E..Stoll, J.. Rau. C. F. Hillenburg. K. C, Kil-loy. W. J.. Drisko. C. L., Wiiliams, R.,Weber. H. P.. Clark. W. Magnusson, I.,Wilker, C & Hujoel. P R < 1990b) Multi-center clinical evaluation of a chairsidemethod for detecting certain peri-odontopathic bacteria in periodontal dis-ease. Journal of Periodontologv 61, 189-196.

Loesche, W J., Giordano, J. & Hujoel, P P.(1990c) The utility of the BANA test formonitoring anaerobic infections due tospirochetes {Treponema denticola) in peri-

Loesche, W J., Giordano, J., Soehren, Hutchinson, R., Rau, C. F, Walsh, LSchork. M. A. (1996) The non-surgtreatment of periodontal patients. OMedicine, Oral Surgery. Oral Pathol81 , 533-543.

Loesche, W. J., Grossman. N. & Giordano(1993) Metronidazole m periodontitis: The effect of patient compliance on trement parameters. Journal of Clinical Podontoiogy 20, 96-104.

Loesche. W J., Lopatin, D. E.. Giordano,Alcoforado. G. & Hujoel. P (1992b) Coparison of the BANA test, DNA proand irrununologicai reagents for their aity to detect anaerobic periodontal inftions due to Porphyromonas gingivaTreponema denticola and Bacteroides sythus. Journal of Clinical Microbiol30, 427^33.

Loesche, W. J.. Lopatin, D. E., Stoll. J., VPoperin. N. & Hujoel, R P (1992a) Coparison of various detection methods perjodontopathic bacteria: can culturbe considered as the primary referestandard? Journal of Clinical Microbio30. 420-426.

Loesche. W. J.. Syed. S. A.. Schmidt. EMorrison. E. C. (1985) Bacterial profilesubgingival plaques in periodontiJournal of Periodontology 56, 447-456.

Lotufo. R. F M., Flynn. J., Chen, C. & SlJ. (1994) Molecular detection of Bacoides forsythus in human periodontiOral Microbiology. Oral Immunology154-160.

Marks. R. G.. Magnasson. I.. Taylor. Clouser, B., Marunjak, J. & Clark. W(1993) Evaluation of reliability and repducibility of dental indices. JournalClinical Periodontology 20, 54-58.

McLaughlin. W S.. Lovat. F M.. & MGregor, I. D. M. (1993) The immediate fect of smoking on gingival fluid floJournal of Clinical Periodontologr 20, 4451 .

Savitt. E. D.. Strzempko, M. N. & VaccaK. K. (1988) Comparison of cultumethods and DNA probe analyses for detection oi .Actinobacillus actinomycecomitans. Bacteroides gingivalis. aBacteroides intermedius in subgingiplaque samples. Journal of Periodontolo59, 431^38.

Simonson, L. G.. Robinson, P. J.. Pranger,J., Cohen, M. E. & Morton, H. E. (19Treponema denticola and Porphyromogingivalis as prognostic markers followperiodontal treatment. Journal of Perionlology 63, 270-273.

Socransky. S. S.. Dibart, S.. Cugini, M. Smith, C. Kent. R. L. Jr. & Haffajee, D. (1996) Biological effects of scahng aroot planing (III) Poor responders. Jourof Dental Research 75 (sp. issue abstr. 9J35.

Watson. M. R., Bretz, W. A. & Loesche. J. (1994) Presence of Treponema dentic

7/28/2019 13461440

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726 Loesehe et al.their parents. Journal of Dental Research73 , 1636-1640.

Williams, R. C , Beck, J . D., & Offenbach-er.S.N. (1996) The impact of new techno-logies to diagnose and treat periodontaldisease. .\ look at the future. J. Ciin Peri-odontol 23, 299-305.

Wolfe, L. E, Anderson. L. A., Sandberg. G.R, Reither, L., Binsfield, C. A., Corinalde-si, G. & Shelburne. C. E. (1992) Bacterialconcentration fluorescence immunoassav

(BCFIA) for detec t ion of periodontopa-thogens in plaque. Journal of Periodonto-logy ki, 1093-1101.Zambon, J . J . , Reynolds, H. S. & Chen, P.(1985) Rapid identification of periodontalpathogens in subgingival dental plaque.Com pariso n of indirect immnnofluor-escence microscopy with bacterial culturefor detection of Baeteroides gingivalis.

Journal of PeriodontoSogv 56 (suppl. 11),32-40 .

Zeger, S. L., Liang, K-.-Y. (1986) Longitnal data analysis for discrete and contous outcomes . Biometr ics 42, 121-130

Address :Walter J. LoescheU niversity of Miehig an Schooi of D entist1 0 1 1 N . U n i v er si t y. A n n A r b or , Mi c h i ga n 4 S 1 0 9 - W7 8U SA

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