JOURNAL OF CLINICAL MICROBIOLOGY, July 1990, p. 1551-1559 0095-1137/90/071551-09$02.00/0 Copyright 1990, Americn Society for Microiology Vol. 28, No. 7 Development of Dignostic Test for Aneroic Periodontl Infections Bsed on Plque Hydrolysis of Benzoyl- DL-Arginine-Nphthylmide WALTER J. LOESCHE,l* WALTER A. BRETZ,' DANIEL KERSCHENSTEINER,2 JANICE STOLL,' SIGMUND S. SOCRANSKY,3 PHILIPPE HUJOEL,' AND DENNIS E. LOPATIN' Deprtment of Biologic nd Mterils Sciences, The University of Michign School of Dentistry, Ann Aror, Michign 48109-10781; 121 Reveille Rod, Wyne, Pennsylvni 190872; nd The Forsyth Dentl Center, Boston, Msschusetts 021153 Received 18 Decemer 1989/Accepted 27 Mrch 1990 Treponem denticol, Porphyromons (Bcteroides) gingivlis, nd Bcteroides forsythus re mong the neroic species frequently ssocited with dult forms of periodontl disese. These orgnisms hydrolyze the synthetic peptide enzoyl-dl-rginine-nphthylmide (BANA), nd such enzyme ctivity cn e detected in the plque nd relted to clinicl disese nd the presence of spirochetes. In this investigtion, the liquid BANA ssy ws compred with commercilly developed BANA ssy which employed pper formt nd which could e red fter 15-min incution. In the pper formt, strips of Whtmn filter pper were impregnted with BANA nd strips of nitrocellulose pper were impregnted with fst lck K slt. Both strips were pplied lengthwise cross pper crd (3 y 5 in. [7.6 y 12.7 cm]). The BANA strip t the ottom ws inoculted with the test smple (pure culture, plque), folded ck so tht it contcted the fst lck strip, nd then incuted for 15 min t 55 C. T. denticol, P. gingivlis, nd B. forsythus lwys gve positive rection, wheres 51 other plque species were lwys negtive. Six Bcteroides nd Cpnocytophg species on occsion hd wek rections. The proportionl greement etween BANA positiveness nd clinicl disese ws similr for oth the liquid nd the pper ssys. The sensitivity, specificity, nd ccurcy reltive to the clinicl stndrd of the liquid ssy were 74, 76, nd 77%, respectively, while those of the pper ssy were 81, 78, nd 80%, respectively. The pper ssy ws significntly ssocited with the presence of either T. denticol or P. gingivlis or oth in the plque smples, with sensitivity of 85%, specificity of 53%, nd n ccurcy of 79%. These findings indicte tht rpid pper ssy for BANA hydrolysis gives dt comprle to those otined with the liquid BANA ssy. Treponem denticol, Porphyromons (Bcteroides) gingivlis (17), nd Bcteroides forsythus re mong the cteril species frequently ssocited with dult forms of periodontl disese (3, 9, 13). These orgnisms re grmnegtive scchrolytic neroic species tht pper to e unique mong the cteri which reside on the tooth surfces in tht they possess n enzyme which hydrolyzes the synthetic peptide enzoyl-dl-rginine-nphthylmide (BANA) (8). This enzyme ctivity is detectle in sugingivl plque smples nd hs een sttisticlly ssocited with the levels nd proportions of spirochetes in the plque (1, 12), with pocket proing depth (6, 12), with clinicl disese t the smpled site (16, 20; W. A. Bretz, D. E. Loptin, nd W. J. Loesche, Orl Microiol. Immunol., in press), nd with the immunologicl detection of T. denticol nd P. gingivlis in the sme plque smples (Bretz et l., in press). The BANA sustrte is the most ccurte of severl synthetic nphthylmide sustrtes in reflecting clinicl disese in untreted periodontl ptients (20). Moreover, the enzymtic ctivity for equl mounts of plque ws higher in disesed ptients thn in cliniclly treted nd mintined ptients (6). In these studies (12, 16; Bretz et l., in press), plque smples were collected from single sites, dispersed, nd diluted nd suitle portions of the plque suspension were * Corresponding uthor. incuted overnight with the BANA sustrte. The color ws developed y the ddition of fst grnet (7). This simple procedure does not lend itself redily to clinicl setting, s the regents must e freshly prepred from stock solutions nd the 18- to 24-h incution does not permit n immedite ppliction of the test results to the mngement of the ptient. In this study, commercilly developed BANA ssy, which employs solid-stte formt nd cn e red fter 15-min incution period, ws compred with the originl BANA ssy. MATERIALS AND METHODS Pure cultures. Pure cultures of P. gingivlis ATCC 33277 nd recent isoltes, strins W nd T, were grown for 4 dys in enriched Trypticse soy roth (BBL Microiology Systems, Cockeysville, Md.) (11). T. denticol ATCC 35405 nd recent isolte ASLM were grown in tryptone-vel hert infusion-yest extrct medium (14) for 5 to 7 dys. The cenls were hrvested y centrifugtion, wshed once in reduced trnsport fluid (10), nd resuspended in reduced trnsport fluid to give finl suspension of out 109 CFU/ml. In experiments determining the detection limits of the BANA ssy, the cell suspensions were diluted 1:5, 1:10, 1:50, 1:100, 1:500, 1:1,000, nd 1:5,000 in reduced trnsport fluid. Smples of 5,ul of the vrious dilutions were pplied directly to the BANA strip contined on the Perioscn regent crd (see elow). Downloded from http://jcm.sm.org/ on Septemer 30, 2018 y guest 1551
1552 LOESCHE ET AL. The following plque species were Americn Type Culture Collection (Rockville, Md.) nd Forsyth Dentl Center reference strins s well s fresh isoltes. These species were Perioscn positive (numer of strins tested): Treponem denticol (3), Porphyromons (Bcteroides) gingivlis (16), nd Bcteroidesforsythus (11). These species were vrile in the Perioscn test: Bcteroides cpillosus (2), Bcteroides denticol (1), Bcteroides orlis (6), Cpnocytophg gingivlis (7), Cpnocytophg ochrce (9), nd Cpnocytophg sputigen (3). These species were Perioscn negtive: Actinocillus ctinomycetemcomitns (12), Actinomyces isrelii (7), Actinomyces neslundii (12), Actinomyces viscosus (6), Bcteroides scchrolyticus (1), Bcteroides ucce (1), Bcteroides denticol (1), Bcteroides grcilis (4), Bcteroides heprinolyticus (1), Bcteroides intermedjus I (7), Bcteroides intermedius Il (6), Bcteroides loescheii (1), Bcteroides melninogenicus (5), Bcteroides oris (1), Bcteroides ureolyticus (1), Bcteroides oulorum (1), Bcteroides zoogleoformns (1), Cmpylocter concisus (4), Cmpylocter sputorum (1), Eikenell corrodens (13), Escherichi coli (2), Fusocterium nucletum susp. nucletum (11), Fusocterium nucletum susp. polymorphum (7), Fusocterium nucletum susp. vincenti (5), Fusocterium periodonticum (1), Hemophilus phrophilus (9), Hemophilus prphrophilus (2), Hemophilus segnis (1), Lctocillus cidophilus (1), Peptostreptococcus micros (7), Propionicterium cnes (5), Selenomons rtemidis (1), Selenomonsflueggeii (1), Selenomons noxi (1), nd Selenomons sputigen (1), Streptococcus constelltus (8), Streptococcus intermedjus (11), Streptococcus mitis (8), Streptococcus morillorum (11), Streptococcus mutns (7), Streptococcus slivrius (10), Streptococcus snguis I (13), Streptococcus snguis II (7), Streptococcus sorinus (4), Streptococcus ueris (5), Treponem pectinovorum (1), Treponem vincentii (1), Veillonell prvul (6), Wolinell cur (2), Wolinell rect (8), Wolinell succinogenes (1). Reference strins were revived from storge in liquid nitrogen, checked for purity, nd confirmed s the designted species y using phenotypic chrcteristics (2, 3). Fresh isoltes were otined from sugingivl plque smples, chrcterized, nd identified s descried previously (2, 3). When multiple strins of species were tested, every isolte ws otined from different suject. A totl of 295 strins representing 57 species were tested, s summrized elow. The orgnisms were grown on Trypticse soy gr pltes supplemented with 5% sheep lood nd incuted t 35C in n tmosphere of 80% N2, 10% H2, nd 10% C02 for 2, 5, or 10 dys. A lrge loopful of growth ws removed from the surfce of the gr pltes with pltinum loop nd plced on the Perioscn test strips. The strips were incuted t 55 C. All isoltes were tested in duplicte on three seprte occsions, i.e., fter 2, 5, or 10 dys of incution. The rection ws scored s strongly positive when lrge re of lue pproximted the deposited cell mss. Wek or questionly positive ws recorded when rections were smll res of lue t loclized spots pproximting the cteril deposit. When discrepncies occurred etween replicte experiments of the sme strin or etween strins of the sme species, the purity of ech strin ws reconfirmed nd the BANA rection ws ressessed. Plque smples. Either suprgingivl or sugingivl plque smples were collected with periodontl curette from single tooth sites in ptients dignosed s hving dvnced periodontitis nd plced immeditely into plstic vil contining 0.1 to 0.5 ml of reduced trnsport fluid. The J. CLIN. MICROBIOL. proing depth of ech smple site ws mesured with thin metl rod tht ws clirted in millimeters ( periodontl proe), nd it ws noted whether the site led upon proing. The clinicin ws then required to stte whether the site ws helthy or disesed, sing this judgement on the proing depth, leeding tendencies, nd tissue ppernce (16). A disesed site ws one which exhiited leeding on proing nd hd proing depth of.4 mm. A helthy site ws one in which there ws no leeding upon proing. Microscopic counts. The plque smples were dispersed for 20 s with vortex mixer, nd 10,ul ws removed for microscopic exmintion. Either 200 orgnisms or the numer of orgnisms in 20 high-power fields (hpf) were enumerted, depending on which event occurred first. The single cells were identified s spirochetes, selenomonds, viriolike motile rods, fusiforms, nonmotile rods, or cocci (11). BANA nlysis. The BANA nlysis ws performed in three formts: the stndrd liquid ssy, solid-stte sndwich ssy, nd solid-stte crd ssy. The protocol for the stndrd ssy hs een descried previously (1, 12) nd will e riefly summrized. A stock solution of BANA (44 mg in 1 ml of dimethyl sulfoxide) ws diluted 1 to 100 in Sorensen uffer to give working solution of 0.67 mm BANA. Portions of 100,ul of this BANA solution were dded to 100,ul of the vrious cteril or plque suspensions contined in well of plstic plte (Minitek; BBL Microiology Systems). The mixture ws incuted overnight, nd then the color ws developed y the ddition of 50,ul of fst grnet solution. The resulting yellow-to-red color ws red in erly studies y eye s negtive (yellow), wekly positive (yellow-ornge), or positive (ornge to red). In susequent studies, the color ws red with n enzymelinked immunosorent ssy (ELISA) reder with 405-nm filter, nd n sornce of less thn 0.10 ws recorded s negtive result. A prototype sndwich technique ws developed in collortion with Orl-B Lortories, Inc. (Redwood City, Clif.) in which Whtmn filter pper ws impregnted with BANA nd nitrocellulose pper ws impregnted with fst lck K slt. Fst lck ws sustituted for the fst grnet ecuse of its superior stility once the color ws developed nd ecuse its lue-lck color, unlike the red-ornge color of fst grnet, is esily distinguishle from the color of lood. Preliminry experiments with P. gingivlis nd T. denticol showed tht the mximl color development occurred when 1:50 dilution of the ove-descried stock BANA solution in 15 mm Tris hydrochloride (ph 8.5) ws dded to the filter pper (pper 903; Schleicher & Schuell, Inc., Keene, N.H.) nd 0.2% fst lck K slt ws dded to the nitrocellulose pper (pure nitrocellulose BA 95; Schleicher & Schuell). In the sndwich ssy, it ws necessry to concentrte the plque suspension. Accordingly, from 50 to 100,u ws removed nd centrifuged for 5 min (microcentrifuge model 235C; Fisher Scientific Co., Pittsurgh, P.). The superntnt fluid ws discrded, nd the pellet ws suspended in 5,ul of phosphte-uffered sline. This suspension ws spotted on the BANA-treted pper. After the BANA pper hd een inoculted, the fst-lck-treted nitrocellulose pper ws moistened with wter nd plced over the BANA pper, nd the ppers were clmped together with pper clips. These sndwich preprtions were incuted for vrious times nd tempertures. In the descried experiments, the preprtions were incuted for 30 min t 45 C, unless stted otherwise. A fint lue or lue-lck color in the fst lck pper ws considered wek positive rection, nd Downloded from http://jcm.sm.org/ on Septemer 30, 2018 y guest
VOL. 28, 1990 DIAGNOSTIC TEST FOR ANAEROBIC PERIODONTAL INFECTIONS 1553 r..j.c5o f )!'. C. ) L-) f\, o Jf )lr ;f ) X(tSf\ if \ )... 1'!._ t........... &,AJA.ÂAlÂJÂ1AfAÂ:&1ÂJA.41. j,1 FIG. 1. Perioscn crd showing positive BANA rections t different cteril concentrtions of P. gingivlis ATCC 33277. Seril dilutions of the strin were lterntely plced on either the upper or the lower BANA-impregnted strip, eginning t sites numered 1, 31, 3, 29, etc. The strip contining fst lck, which is positioned etween the drwings of the teeth, ws moistened with wter, nd then the BANA strips were folded up on the fst lck strip. The folded crd ws plced in the metllic clip shown t the ottom nd then incuted in het lock t 55 C for 15 min. The positive rections were oserved on the fst lck strip, with the strongest rection t site 1. distinct lue or lue-lck color ws considered positive. This ssy ws le to detect 5 to 10 ng of trypsin per,ul (ovine pncretic trypsin type III; specific ctivity, pproximtely 11,000 BAEE units/mg of protein; Sigm Chemicl Co., St. Louis, Mo.). The crd ssy ws n extension of the sndwich technique nd represented commercil product (Perioscn) tht ws developed in conjunction with Orl-B Lortories. The BANA- nd fst-lck-impregnted ppers were pplied s strips tht rn lengthwise cross crd (3 y 5 in. [7.6 y 12.7 cm]) (Fig. 1). The BANA strips t the ottom could e folded up so tht they contcted the fst lck strip. This contct ws mintined y metllic clip into which the folded crd could e inserted. This crd formt ws studied to determine the sensitivity of the ssy for vrious pure cultures. The pure cultures were concentrted y centrifugtion, nd 5 IlI of ech culture ws slowly pplied s discrete spot on the BANA strip. The fst lck strip ws moistened with distilled wter, nd then the BANA strip ws folded up onto it nd held in plce y the clip (Fig. 1). In the descried experiments, the BANA crd ws incuted for 15 min t 55 C unless stted otherwise. The crds were red s follows: negtive, sence of lue or lue-lck color; wekly positive, fint lue or lue-lck color; positive, discrete lue-lck color. Vrious pure cultures of T. denticol nd P. gingivlis, plque suspensions, nd trypsin stndrd were inoculted in this fshion nd incuted for vrious times nd tempertures. Pure cultures were grown to the erly sttionry phse, concentrted y centrifugtion, suspended in phosphte-uffered sline, nd diluted in phosphte-uffered sline. A 5-,u smple of ech of three dilutions per orgnism ws spotted long with trypsin stndrd solution onto single crd. Enough crds were inoculted to incute duplicte crds for 5, 15, 30, nd 60 min t 25, 35, 45, 55, 65, or 75 C. The dilutions chosen included cell count known to yield positive rection in the sndwich technique, cell count tht ws 8- to 10-fold higher thn this positive control, nd cell count tht ws 500-fold lower, which served s negtive control. The trypsin stndrd ws tested t 10 nd 1.3 ng/,ul. ELISA. The lower regent strip contining the plque ws removed from the Perioscn crd, nd then the strip ws cut in hlf nd ech hlf ws incuted in solution of highly specific hyperimmune rit ntiodies to either P. gingivlis or T. denticol. The strips were then wshed in phosphte-uffered sline-tween uffer nd then incuted in lkline phosphtse-conjugted got nti-rit immunogloulin G. The immune complexes were reveled fter incution with BCIP/NBT phosphtse sustrte (Kirkegrd nd Perry Lortories, Inc., Githersurg, Md.). This ELISA procedure will detect out 5 x 104 CFU of either T. denticol or P. gingivlis, which is out 10-fold more sensitive thn the Perioscn. The specificity of the ntiod- Downloded from http://jcm.sm.org/ on Septemer 30, 2018 y guest
1554 LOESCHE ET AL. TABLE 1. Reltionship etween BANA hydrolysis s detected y fst grnet (liquid) nd fst lck (pper) nd the levels of spirochetes in plque smples Test result No. of spirochetes/hpfn with: Fst grnet Fst lck Negtive 1.6 + 3.5 (43) 1.8 + 3.6 (44) Wekly positive 14.1 + 5.4 (18) 8.8 + 10.8 (5) Positive 30.6 3.7c (38) 27.2 + 3.4" (50) ANOVAd P 0.0002 P = 0.0145 Averge + stndrd error. The numer of plque smples is shown in prentheses. Vlues re significntly different from ech other. c Vlue significntly different from other vlues in the sme column. d Two-fctor ANOVA with one rndom fctor eing the ptient. ies nd the detils of the protocol re descried elsewhere (Bretz et l., in press). The ELISA rections were grded s negtive, wekly positive, or positive. Sttistics. Ech of the dt sets, i.e., enzymtic, clinicl, nd microscopic, ws determined independently nd then compiled for sttisticl nlysis. A two-fctor nlysis of vrince (ANOVA) (P. Hujoel, L. Moulton, nd W. Loesche, J. Periodontl Res., in press) ws performed on dt otined with the Perioscn, with the liquid BANA ssy, nd with the sndwich formt ssy. In this nlysis, the independent fctor ws the BANA ssy or ELISA (negtive, wekly positive, or positive) (fixed fctor), the ptient ws rndom fctor, nd the dependent vriles were the vrious plque prmeters under investigtion. The sensitivity, specificity, nd ccurcy of the Perioscn test results were determined reltive to those of the ELISA s cteril stndrd for T. denticol nd P. gingivlis. All these estimtes were clculted y the correlted inomil model, which tkes into ccount the fct tht sites within ptient re correlted nd llows for heterogeneous numer of responses within ptient (Hujoel et l., in press). Similrly, the oserved proportionl greement of the Perioscn, the liquid BANA ssy, nd the sndwich ssy with the clinicl sttus of the smpled site nd the oserved proportionl greement etween the Perioscn nd the liquid BANA ssy were clculted y the correlted inomil model. RESULTS Preliminry experiments hd shown tht procedure in which inocul of P. gingivlis nd T. denticol were sndwiched etween filter pper impregnted with BANA nd nitrocellulose pper impregnted with fst lck gve rise to lue or lue-lck color in the nitrocellulose pper. The color ws permnent nd could e developed within 15 to 60 min, depending on the incution temperture (etween 37 nd 60 C). A temperture of 45 C nd 30-min incution period were chosen for n experiment in which suprgingivl nd sugingivl plque smples were nlyzed y oth the liquid ssy with fst grnet s the color developer nd the pper sndwich ssy with fst lck s the developer (Tle 1). Both the liquid nd sndwich ssys yielded comprle informtion reltive to the levels of spirochetes tht were present in microscopic hpf (one orgnism per hpf is equivlent to pproximtely 5 x 106 cells per ml). A positive rection ws ssocited with out 27 to 30 spirochetes per hpf, while negtive rection ws ssocited with fewer thn 2 spirochetes per hpf. Of the 56 positive plques with the fst grnet developer, 18 were judged s wekly positive, J. CLIN. MICROBIOL. TABLE 2. Comprison of liquid nd pper ssys for BANA hydrolysis in sugingivl plques removed from periodontlly helthy nd disesed sites Assy % Positive % Negtive Totl % Agreend site plques plques plfqe mentl Liquid (fst grnet) 77 Disesed 74 26 46 (100) Helthy 21 79 14 (100) Totl no. of plques 38 22 60 Pper (fst lck) 80 Disesed 80 20 46 (100) Helthy 14 86 14 (100) Totl no. of plques 39 21 60 Oserved proportionl greement estimtes were clculted y the correlted inomil model (Hujoel et l., in press). wheres only 5 of the 55 positive plques in the fst lck rection were judged s wekly positive. As the intensity of color ws determined sujectively y visul exmintion, this suggested tht the viewer ws etter le to recognize positive rections with the fst lck developer in the sndwich ssy. The liquid ssy hs een shown to gree significntly with the ssessment y the clinicin of helth or disese in the smpled tooth site (16; Bretz et l., in press). In the present experiment, the proportionl greement etween BANA positiveness nd disese ws similr for oth the liquid nd the pper ssys (Tle 2). The sensitivity (true positive), specificity (true negtive), nd ccurcy [(true positive + true negtive)/totl numer] reltive to the clinicl stndrd (disese eing positive, helth eing negtive) of the liquid ssy were 74, 76, nd 77%, respectively, while those of the pper ssy were 81, 78, nd 80%, respectively. These percentges were slightly lower thn the vlues shown in Tle 2, which were clculted directly from the numer of plques exmined without tking into ccount tht some of the plques were clustered within ptients (Hujoel et l., in press). Thus, the pper ssy ws essentilly identicl to the liquid ssy. Since the pper ssy could e performed in 30 min, these results suggested tht it would e possile to otin BANA test result during the sme clinicl visit in which the plque ws collected. The pper ssy ws further modified to fcilitte its clinicl use. The wkwrd sndwich procedure ws replced y the Perioscn crd in which the plque could e plced on the BANA strip nd then folded up to mke contct with the fst lck strip. The drwings of the teeth were dded to fcilitte interprettion of the results (Fig. 1). The optiml temperture of incution nd length of incution were determined with Americn Type Culture Collection strins of T. denticol nd P. gingivlis. The hevy cell suspension contining 62 x 106 CFU of T. denticol per 5,ul reched mximum color intensity fter 30 min of incution t 35 C ut needed only 5 min of incution t 55 C (Fig. 2). The cell suspension contining 5 x 106 CFU/5,ul reched its mximum color intensity fter 15 min of incution t 55 C. This level, which pproximted the level of T. denticol seen in most plque smples from disesed tooth site, exhiited temperture optimum t 55 C. The light cell suspension of T. denticol, i.e., 1.5 x io4 CFU/5,ul, ws negtive t ll times nd t ll tempertures. The BANA enzyme in P. gingivlis ppered to e more Downloded from http://jcm.sm.org/ on Septemer 30, 2018 y guest
VOL. 28, 1990 DIAGNOSTIC TEST FOR ANAEROBIC PERIODONTAL INFECTIONS 1555 E 0.2 e 0o m. 0 1-0o 2 3 2 1 02 25 35 45 Temperture 55 65 75 FIG. 2. Br grph showing intensity of BANA color rection s function of incution temperture ( C), time of incution, nd numer of cells of T. denticol ATCC 35405. het stle thn the T. denticol enzyme, s the mximum color intensity with the hevy cell suspension occurred within 5 min t 65 C (Fig. 3). At levels of 9 x 106 CFU/5, the color ws less intense nd did not increse with either the length or temperture of incution eyond tht seen fter 15 min of incution t 45 C. Trypsin (10 ng/,ul) ws positive t ll tempertures fter 15 min of incution (dt not shown). These findings led to selection of 15 min t 55 C s the stndrd protocol in which we evluted the detection limits E 0 og c. 2 o 0 o A 3-2- * 5 min 15 min 30 min E60 min of the Perioscn crd for two strins of T. denticol nd three strins of P. gingivlis. The cell count tht ws one fivefold dilution elow the lst wekly positive BANA rection ws selected s the detection limit. Vrition ws noted etween different strins of the sme species, s well s within different replictions of the sme strin (Tle 3). On the verge, out 1 x 106 CFU of T. denticol were required to give wekly positive rection, wheres out 2 x 105 CFU of P. gingivlis were required. () 75 x 10 ()9x 10 Downloded from http://jcm.sm.org/ on Septemer 30, 2018 y guest *o E 1 M-A 25 35 45 55 65 75 Temperture (OC) FIG. 3. Br grph showing intensity of BANA color rection s function of incution temperture, time of incution, nd numer of cells of P. gingivlis ATCC 33277. I
1556 LOESCHE ET AL. TABLE 3. Detection limits for Perioscn test for T. denticol nd P. gingivlis Strin 105 CFU Rnge (10') No. of replictions T. denticol ATCC 35405 ASLM 8.4 ± 12.0 21.8 ± 15.4 0.2-57.5 6.6-49.6 21 8 P. gingivlis ATCC 33277 3.3 ± 2.9 0.8-0.0 12 T 1.5 ± 1.3 0.3-5.2 12 W 1.2 ± 0.8 0.05-2.7 12 Men + stndrd devition. A lrge pnel of reference strins ws then evluted for its ility to give BANA rection in the Perioscn formt. Three ptterns were oserved. Only strins of P. gingivlis, B. forsythus, nd T. denticol exhiited consistent positive rections (see Mterils nd Methods). Thirty strins of these species, tested on t lest three occsions, uniformly yielded positive rections. Most species gve BANAnegtive rection. Thus, 244 strins representing 51 species did not exhiit ny BANA-positive rections. Two spirochetes, T. vincentii nd T. pectinovorum, s well s four lck-pigmented cteroides species, i.e., B. intermedius I, B. intermedius II, B. loescheii, nd B. melninogenicus, were uniformly BANA negtive. A vrile rection ws oserved with certin Bcteroides nd Cpnocytophg species in which 36 of 152 seprte determintions were positive or wekly positive (see Mterils nd Methods). The inconsistent rections of this group of strins led to the following experiments. Initilly, ll cultures were confirmed for purity on the sis of colonil nd microscopic exmintion nd iochemicl tests. Thus, inconsistency could not e ttriuted to mixed cultures or misidentifiction. The reltionship of time nd incution of the test strins on lood gr pltes with the susequent positive or negtive test rection ws exmined, ut no cler reltionship could e estlished. Cells grown in roth nd hrvested y centrifugtion were no more consistent in their rection thn were cells hrvested from gr surfces. Even the use of extremely lrge cell msses (s much s could e physiclly plced on the strip) did not result in consistent positive rections for these test strins. In susequent experiments, sugingivl plques were removed from periodontlly involved teeth nd, fter dispersion, equl portions were evluted y the liquid BANA nd Perioscn ssys. The mjority of the plques were Perioscn negtive, nd this coincided with the inility of the plque to hydrolyze BANA in the liquid ssy nd with the presence of low levels nd proportions of spirochetes in the TABLE 5. J. CLIN. MICROBIOL. Agreement etween Perioscn BANA results nd liquid BANA results Liquid BANA % Perioscn % Perioscn Totl result positive' negtive plques Positive 88 18 57 Negtive 12 82 127 Totl plques (%) 34 (100) 150 (100) 184 Oserved proportionl greement of 83% ws clculted y the correlted inomil model (Hujoel et l., in press). The kpp vlue ws 0.58. Proportion of plques. plque smples (Tle 4). When the dt were nlyzed y two-fctor ANOVA, there were highly significnt differences mong the Perioscn scores nd (i) BANA hydrolysis, (ài) totl spirochetes in the smple, (iii) spirochetes per hpf, nd (iv) percent spirochetes (Tle 4). The clerest differences were oserved with the BANA hydrolysis nd with the totl spirochetes in the smple, s their vlue t ech Perioscn score ws significntly different from their vlues t the other Perioscn scores. This ws to e expected if the Perioscn nd liquid BANA ssy were mesuring the sme enzyme ctivities nd if most of this ctivity ws due to the spirochetl, i.e., T. denticol, lod of the smple (12). The liquid BANA nd the Perioscn BANA results were relted to ech other with n 83% greement (Tle 5). The min difference etween the tests ws the greter numer of positive rections found in the liquid ssy, i.e., 57 versus 34 in the Perioscn ssy. This could e ttriuted to the higher numer of wekly positive results detected in the liquid ssy, i.e., 31 versus 16 for the Perioscn ssy. This suggested tht the overnight incution period for the liquid BANA ws permitting the detection of smll numers of BANA-positive orgnisms tht pprently could not e detected during the 15-min incution used in the Perioscn ssy. The inoculum used for oth BANA ssys in these experiments ws only frction of the plque suspension nd not the entire plque smple. In prctice, however, the entire plque smple would e used for the Perioscn ssy. We next evluted the reltionship etween the Perioscn results nd clinicl sttus when the entire plque smple ws plced on the BANA strip. These findings were lso relted to the presence of T. denticol nd P. gingivlis in the plque s ssessed y n ELISA. The Perioscn results were significntly relted to the presence or sence of either T. denticol or P. gingivlis or oth in the plque smples (Tle 6) with sensitivity of 85%, specificity of 53%, nd n ccurcy of 79%. The Perioscn results were lso in greement with the clinicl sttus of the smpled site (Tle Downloded from http://jcm.sm.org/ on Septemer 30, 2018 y guest TABLE 4. Reltionships mong Perioscn test, BANA hydrolysis, nd spirochete profile of plque smples Perioscn No. of BANA hydrolysis Spirochete profile result plques (A405) Totl in smple (105) No./hpf % Negtive 150 0.08 ± 0.009 2.2 ± 0.9 0.1 ± 0.1 5.3 ± 0.8 Wekly positive 16 0.28 ± 0.03 13.2 ± 2.9 0.7 ± 0.4 16.8 ± 2.5 Positive 18 0.51 ± 0.03 28.3 ± 2.8 2.6 ± 0.4 21.8 ± 2.3 ANOVAC P = 0.0001 P = 0.0001 P = 0.0001 P = 0.0001 Averge ± stndrd error. Vlues significntly different from other vlues in sme column. C Two-fctor ANOVA with one rndom fctor eing the ptient.
VOL. 28, 1990 DIAGNOSTIC TEST FOR ANAEROBIC PERIODONTAL INFECTIONS 1557 TABLE 6. Reltionship etween Perioscn nd ELISA results for oth T. denticol nd P. (Bcteroides) gingivlis Perioscn ELISA Totl result % Negtive % Positive plques Negtive 56 15 41 Positive 44 85 139 Totl plques (%) 36 (100) 144 (100) 180 Sensitivity (85%), specificity (53%), nd ccurcy (79%) estimtes were clculted y the correlted inomil model. These differ slightly from vlues clculted y using the numer of sites. 7). If we use the clinicl judgment s the reference stndrd, then the Perioscn test hd sensitivity of 86%, specificity of 40%, nd n ccurcy of 74%. DISCUSSION The liquid BANA ssy hs een shown to relily indicte the presence of high levels of spirochetes (12) ndlor T. denticol nd/or P. gingivlis (Bretz et l., in press) in the plque smple. Since B. forsythus is lso BANA positive (21), the Perioscn test cn give informtion on this orgnism. The test will not tell which of these orgnisms is present, ut since they il re neroic species, it should enle the clinicin to dignose n neroic infection, nd such dignosis could e useful for the tretment nd mngement of the periodontl disese of the ptient. For instnce, the liquid BANA test ws used to dignose n neroic infection which led to the use of metronidzole nd n improvement in the periodontl helth of the ptient reltive to control ptients (W. J. Loesche nd P. Hujoel, in N. W. Johnson, ed., Mrkers of Disese Susceptiility nd Activity for Periodontl Disese, in press). Also, positive BANA test fter scling nd root plning ws ssocited with high proportions of spirochetes, suggesting tht the tretment rendered ws not dequte to suppress or eliminte the neroic infection. This ws supported y the oservtion tht teeth with BANA-positive plques t the conclusion of scling nd root plning, s opposed to teeth with BANA-negtive plques, lost significntly more ttchment during the yer following tretment (W. L. Loesche, J. Giordno, nd P. Hujoel, J. Dent. Res. 69:354, 1990). This ckground led us to develop the BANA ssy in formt tht would e simple, quick, nd relile for use y the dentist t chirside. The present findings indicte tht the solid-stte pper ssys for BANA hydrolysis tht hve een developed give dt comprle to those of the liquid BANA ssy. The prototype sndwich method, the Perioscn crd formt, nd the liquid ssy were significntly relted to the TABLE 7. Reltionship etween Perioscn BANA results nd clinicl sttus of smpled site' ~~~~~~~~~~~plques Clinicl Perioscn Totl sttus sttus o% Negtive % Positive pu Helthy 40 60 47 (100) Disesed 16 84 133 (100) Totl plques 40 140 180 Oserved proportionl greement of 73% ws otined y jckknife estimtion procedures, tking into ccount the correlted nture of sites (Hujoel et l., in press). spirochete levels in the plque (Tles 1 nd 4). The liquid nd oth pper ssys gve comprle informtion reltive to the clinicl sttus of the smple tooth site (Tles 2 nd 7), with n ccurcy of 77% for the liquid BANA, 80% for the sndwich BANA (Tle 2), nd 74% for the Perioscn BANA (Tle 7). Other dignostic tests for the periodontl pthogens re eing evluted in vrious lortories. An immunofluorescence procedure employing monoclonl ntiodies to P. gingivlis hd n ccurcy of 69% when relted to the disese sttus of the ptient (22). The detection of P. gingivlis, B. intermedius, nd A. ctinomycetemcomitns y DNA proes hd n ccurcy of 69% when relted to whether the plque cme from smple site with.6-mm proing depth (15). The ccurcies otined with ll three formts of the BANA ssy, i.e., 74 to 80%, re in the sme rnge. However, the criteri for disese ssessment in ech of these studies were different, so tht cross comprisons etween the studies my not e vlid. The Perioscn test could e positively ssocited with the presence of P. gingivlis nd T. denticol in the plque smples with sensitivity of 85% (Tle 6). The specificity of 53% would indicte tht other BANA-positive orgnisms re present in the plque. Among the other species tested with the Perioscn, only B. forsythus nd the Bcteroides nd Cpnocytophg species listed in Mterils nd Methods were le to give positive or wek rections. Thus, it is possile tht these orgnisms re present in plques tht re low in or devoid of T. denticol nd P. gingivlis nd therey ccount for these Perioscn-positive findings. Note tht the in vitro testing of the 60 sugingivl species listed in Mterils nd Methods ws weighted to the detection of positive results, i.e., mssive numers of cells were plced on the test strips. The numer of cells ws t lest s lrge s tht otined y complete removl of plque from deep periodontl pocket. Since plque smple usully hrors mixture of species, the numer of cells of the species exmined in this study should hve een s high s, or higher thn, tht present in plque smples. Thus, if the in vitro dt cn e extended to in vivo systems, positive clinicl test is likely to e due to cells of only limited numer of species. The inconsistent rections oserved for six species deserve dditionl comment. The rections differed in terms of their intensities nd their consistencies. The presence of lrge numers of cells of T. denticol or P. gingivlis or B. forsythus uniformly resulted in lrge lue res pproximting the site of the test inoculum. The inconsistent rections, however, were never s lrge nd, when they occurred, were often confined to one mrgin of the inoculum. The reson for this pttern is not cler, ut it could indicte tht the BANA-hydrolytic enzyme is not consistently produced y the cells or tht the inconsistent species consistently produced this enzyme ut t levels which were mrginl for the sensitivity of the ssy, even when huge msses of cells of tht species were inoculted. These considertions suggest tht B. forsythus nd possily other yet to e identified BANA-positive species, nd not the BANA-vrile species, were responsile for the Perioscn-positive findings in the sence of detectle P. gingivlis nd T. denticol. The most pprent difference etween the liquid nd the pper ssys ws the higher numer of BANA-positive results tht were found in the liquid ssy (Tle 5). This ws ssocited with higher numer of wekly positive results otined with the liquid ssy compred with the pper ssy, i.e., 31 versus 16, nd suggested tht the overnight Downloded from http://jcm.sm.org/ on Septemer 30, 2018 y guest
1558 LOESCHE ET AL. incution period provided enough time for low levels of BANA-positive orgnisms in the plque smples to hydrolyze detectle levels of BANA. These low levels cn e estimted, with some cution, to e out 106 CFU for T. denticol nd out 105 CFU for P. gingivlis. The cution reflects the high degree of vriility encountered when pure cultures of T. denticol nd P. gingivlis were repetedly ssyed for BANA ctivity (Tle 3). This vriility ws gretest with T. denticol, s indicted y the lrge stndrd devitions recorded. These findings indicte tht while these species were consistently BANA positive, the level of enzyme ctivity could e vrile, therey mking it difficult to provide definite detection limit vlues for these orgnisms in the plque smples. The issue of detection limits rises fundmentl questions s to how dignostic test for vrious puttive periodontl pthogens needs to e designed. Since mny of these orgnisms, such s the spirochetes nd T. denticol (9, 18), B. intermedius (3, 19), B. forsythus (3, 5), nd P. gingivlis (5), cn e found in low numers in mny plque smples in the sence of clinicl disese, dignostic test(s) with low detection limit would often yield positive rections in the presence of clinicl helth (Loesche nd Hujoel, in press). If, however, the presence of the puttive pthogen plces the individul tooth or ptient t risk for periodontitis, s hs een suggested for P. gingivlis nd A. ctinomycetemcomitns (4, 19), then test with low detection limits would e preferred. This issue concerning detection versus threshold limit for the puttive pthogen cn e evluted y exmining the reltive ilities of sensitive tests (in the cteriologicl mening), like DNA proes nd ntiodies, nd less sensitive tests, like the BANA hydrolysis, to reflect clinicl disese. In this regrd, we screened young children (verge ge, 6 yers) for the presence of T. denticol nd/or P. gingivlis using the Perioscn ssy nd ELISA. Most children were detectly colonized with T. denticol nd/or P. gingivlis s determined y the ELISA, ut only limited numer were colonized s determined y the BANA ssy (M. R. Wtson, W. A. Bretz, nd W. J. Loesche, J. Dent. Res. 68, 1989). The level of coloniztion y BANA-positive orgnisms ut not ELISA-positive orgnisms could e significntly ssocited with history of periodontl disese in household memer. When mothers of BANA-positive nd BANA-negtive children were exmined cliniclly for periodontl disese, the mothers of the BANA-positive children hd significntly more periodontl disese thn the mothers of BANA-negtive children (M. R. Wtson, P. Hujoel, nd W. J. Loesche, unpulished dt). This suggests tht it is more useful to hve dignostic tests tht detect certin threshold levels thn tests tht detect only coloniztion. The present findings indicte tht solid-stte pper ssy for BANA hydrolysis gives dt comprle to those otined with the liquid BANA ssy. In ddition, the pper ssy hs certin dvntges tht would fcilitte its clinicl use. Thus, result could e otined during the sme clinicl visit in which the plque smple ws tken, therey enling the clinicin to initite suitle therpy. Also, since the fst lck dye gives stle color, the Perioscn crd could e entered into the permnent tretment file of the ptient. ACKNOWLEDGMENT This investigtion ws supported y grnt from Orl-B Lortories, Inc., Redwood City, Clif. J. CLIN. MICROBIOL. LITERATURE CITED 1. Bretz, W. A., nd W. J. Loesche. 1987. Chrcteristics of trypsin-like ctivity in sugingivl plque smples. J. Dent. Res. 66:1668-1672. 2. Dzink, J. L., C. Smith, nd S. S. Socrnsky. 1984. Semiutomted technique for identifiction of sugingivl isoltes. J. Clin. Microiol. 19:599-605. 3. Dzink, J. L., S. S. Socrnsky, nd A. D. Hffjee. 1988. The predominnt cultivle microiot of ctive nd inctive lesions of destructive periodontl diseses. J. Clin. Periodontol. 15: 316-323. 4. Genco, R. J. 1987. Highlights of the conference nd perspectives for the future. Proceedings of the 7th Interntionl Conference on Periodontl Reserch. J. Periodontl Res. 22:164-171. 5. Gmur, R., J. R. Stru, nd B. Guggenheim. 1989. Prevlence of Bcteroides forsythus nd Bcteroides gingivlis in sugingivl plque of prosthodonticlly-treted ptients on short recll. J. Periodontl Res. 24:113-120. 6. Guserti, F. A., S. A. Syed, T. Hofmnn, nd N P. Lng. 1986. Dignostic methods for the ssessment of potentil periodontl disese ctivity: enzymtic ctivities of cteril plque nd their reltionship to clinicl prmeters, p. 165-174. In T. Lehner nd G. Cimsoni (ed.), The orderlnd etween cries nd periodontl disese III. Grune & Strtton, Inc., Orlndo, Fl. 7. Lughon, B. E., S. A. Syed, nd W. J. Loesche. 1982. API-ZYM system for identifiction of Bcteroides sp., Cpnocytophg sp., nd spirochetes of orl origin. J. Clin. Microiol. 15:97-102. 8. Loesche, W. J. 1986. The identifiction of cteri ssocited with periodontl disese nd dentl cries y enzymtic methods. Orl Microiol. Immunol. 1:65-70. 9. Loesche, W. J. 1988. The role of spirochetes in periodontl disese. Portside Symp. Adv. Dent. Res. 2:275-283. 10. Loesche, W. J., R. N. Hockett, nd S. A. Syed. 1972. The predominnt cultivle flor of tooth surfce plque removed from institutionlized sujects. Arch. Orl Biol. 17:1311-1326. 11. Loesche, W. J., S. A. Syed, E. C. Morrison, G. A. Kerry, T. Higgins, nd J. Stoll. 1984. Metronidzole in periodontitis. I. Clinicl nd cteriologicl results fter 15 to 30 weeks. J. Periodontol. 55:325-335. 12. Loesche, W. J., S. A. Syed, nd J. Stoll. 1987. Trypsin-like ctivity in sugingivl plque: dignostic mrker for spirochetes nd periodontl disese? J. Periodontol. 58:266-273. 13. Moore, W. E. C. 1987. Microiology of periodontl disese. J. Periodontl Res. 22:335-341. 14. Oht, K., K. K. Mkinen, nd W. J. Loesche. 1986. Purifiction nd chrcteriztion of n enzyme from Treponem denticol cple of hydrolyzing synthetic trypsin sustrtes. Infect. Immun. 53:213-220. 15. Svitt, E. D., M. N. Strzempko, K. K. Vccro, W. J. Peros, nd C. K. French. 1988. Comprison of culturl methods nd DNA proe nlysis for the detection of Actinocillus ctinomycetemcomitns, Bcteroides gingivlis nd Bcteroides intermedius in sugingivl plque smples. J. Periodontol. 59:431-438. 16. Schmidt, E. F., W. A. Bretz, R. A. Hutchinson, nd W. J. Loesche. 1988. Correltion of the hydrolysis of enzoyl-rginine nphthylmide (BANA) y plque with clinicl prmeters nd sugingivl levels of spirochetes in periodontl ptients. J. Dent. Res. 67:1505-1509. 17. Shh, H. N., nd M. D. Collins. 1988. Proposl for reclssifiction of Bcteroides scchrolyticus, Bcteroides gingivlis, nd Bcteroides endodontlis in new genus, Porphyromons. Int. J. Syst. 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VOL. 28, 1990 DIAGNOSTIC TEST FOR ANAEROBIC PERIODONTAL INFECTIONS 1559 1984. Dignostic potentil of chromogenic sustrtes for rpid detection of cteril enzymtic ctivity in helth nd disese ssocited plques. J. Periodontol. Res. 19:618-621. 21. Tnner, A. C. R., M. N. Strzempko, C. A. Belsky, nd G. A. McKinley. 1985. API ZYM nd API An-Ident rections of fstidious orl grm-negtive species. J. Clin. Microiol. 22:333-335. 22. Zmon, J. J., H. S. Reynolds, P. Chen, nd R. J. Genco. 1985. Rpid identifiction of periodontl pthogens in sugingivl dentl plque. Comprison of indirect immunofluorescence microscopy with cteril culture for detection of Bcteroides gingivlis. J. Periodontol. 56(Suppl. 11):32-40. Downloded from http://jcm.sm.org/ on Septemer 30, 2018 y guest