Methodology

Comparison of two screening tests forStreptococcus mutans and evaluation oftheir suitability for mass screenings andprivate practice

Nt'.wtiRUN, T, MATstiKiino, GHARLRS T, HOOVER, RICHARD G, GRAVES*, ALBERT T, BROWN*, JUDITH

A. DISNEY* AND HARRY M, BOHANNAN*University of Calijornia San Fr<ttteiseo, San Franeiseo. California: *.-\nteriean Ftind for Dental Health: attd *Unt!iersily oJ Kentucky. Lexinglon,Kentucky, t^SA

Newbrun E, Malsnkube) T, He)over GI, Grave\s RG, Brown ,'VT, Disney J,A, Bohannan HM:Gomparison of Iwo sereening test,s for Sireptoeoeens mulans and evaluation ol their .suitability forma.ss .sereenings and j^rivale jiraeliee, Gommeinity Dent Oral Epidemiol 1984; 12: 325 31,

Abslrael Twe) simple niierobioleigieal tesls feir epianlilaling salivat-y Streptoeoeens mutans levelswere e-e)niparecl wilh eaeh other and e\-aliialed for suitability for mass sereenings and privatepraeliee. Beitli lesls use mitis salivarius medium wilh bacitraeiii (MSB) and are selective for S,mutans. One tesi estimates eolonies grown on agar (MSBA) and lhe other estimates colonies grownin broth thai adhere lo glass (MSBB), Bolh are scored trom 1 (lowest) to 5 (highest), Ghildren(293) ill grades 5 and 6 in Tallahassee, Florida (nontluoridaled) were tested for S, mutans levels,Seores of ehildren l)\ be)lli te-sl,s were signifieantly similar {%'-, regression and Kappa statistiealanalysis), Gori-elalie)ii e-e)eHieienls (regression) between scores and DMFS increments of the previous4 yr were 0,35 (MSBA) and 0,26 (MSBB), Be)lh tesls were vec-y good iu identifying children withlow caries incremenls, but ])osilive scores did not correlate well wilh high caries increments. Samplingand inlerpi-eting took 2 min (MSBA) and 1 min (MSBB), Gosl of MSBA/ehild was SL15, andMSBB/child $1,25, These tests are eee)nomieal and suitable for nia,ss sereenings to identify low riskpopulalions who do not require preventive Irealment,

Key words: denial eaiies, aelivily; denial caries, incremenls; ma,ss screenings; Slreplococeus mulans,

E, Newbrun, Division of Oral Biology, Department of Stomatology, HSW-604, Uni\'ersily of Galifor-nia San l''raneisee), ,San I'Vancisce), Galifornia 94143, USA,

Accepled for pcil)lie-alie)n 26 November 1983.

M a n y tests of caries susceptibility have been pro-posed, evaluated and used by both clinicians andresearch workers (1-6), In longitudinal trials, va-rious eharaeteristies of the sttidy subjeets have beenexamined as potential |iredictors of caries incre-ment . These characteristics have included host fac-tors, such as the lluoride content of enatnel and theflo-w rate and buffering capacity of saliva; composi-t ion of the oral microflora, particularly the presenceof Streptococcus mutans and lactobaeilli; andpatients' diet histories. These assessments have metwith dilferent degrees of success, as measured by theassociated coellicient of determination (R^), The

one characteristic most consistently correlated withcaries increment has been the initial DMFS (de-cayed, missing or filled tooth surfaces) score (7), Ifthe past caries experience is considered on a hier-archical basis, it can be an even more effectivepredictor of future caries activity (8, 9),

There are serious drawbacks to using past cariesexperience as a predictor of future caries activity inthe ideutification and seleetion of high risk popula-tions for preventive treatment. The most obviousdisadvantage is that eousiderable caries will havealready occurred in this population. Secondly, thismethod is not applicable to the very young, when

326 NEWBRUN ET AL.

preventive intervention is desirable. Finally, thismethod of identifieation requires professional dentalexaminations.

An ideal earies predictive test should (a) be simpleenough to be performed by nondental personnel;(b) be reproducible; (c) anticipate the onset ofcaries; (d) be inexpensive; and (e) be applicable ona mass basis as well as in the dental oflice. Wecompared two simple microbiological methods ofquantitating S, mutans in saliva for their correlationwith past caries increments and their practicalityfor mass screenings. We also compared the eost ofeach of these tests, based on materials and staff time.These tests were the S, mutans screening test, whichuses a tongue blade for sampling and inoeulatingon mitis salivarius baeitracin agar (MSBA) (10);and the S, mutans adherence test, which uses mitissalivarius baeitracin broth (MSBB) (11, 12).

MATERIAL AND METHODSPopulation We studic;d 299 5t.h and 6th grade ehildren attendingsix public schools in Tallahassee, Florida (a nontlnoridated e:ity),who had been parlicipating as a eontrol populalion in a NationalPreventive Dentistry Demonstration Program, Garies incrementsover the previous 4 yr were available lor 293 of these ehildren.Methods and criteria used in examining for dental caries arereported elsewhere (13), Beeause some ehildren were absent theday of the test or unable to provide saliva for both tests, MSBAdata were obtained for 298 ehildren and MSBB data for 293,Most of the ehildren (92%) had been using lluoride-eonlainingdentifriees.

Salivary sampting - Sampling by the two methods was done ingroups of five children at a time. First, unstimulated whole salivawas expeetorated for the MSBB method, and then eaeh ehildehewed a small ( ~ 1 g) piece of parallin wax for I min to stimulatesaliva for the MSBA method,

MSHA - The MSBA method was developed for use wilh largenumbers of sehoolehildren and avoids the necessity of colle-ctiiigsaliva, Eaeh subjeet ehewexl a piece of paraffin wax for 1 min todisplace plac|ue miercwrganisms, thereby inereasing their pro-portions in the saliva. The subjeel was then given a sterile tongueblade to rotate in the mouth 10 times, so thai bolh sides of thetongue blade were thoroughly inoeulatc'd by the salivary ne)ra.Both sides of the tongue blade were then pressed on to an MSBagar in a disposable eontaet petri dish (RODAG plate). Theplate;s were plaeed into plaslie bags containing expired air uutilineubation at 37°G in 85% N2, 10% H2 and 5% GO'i for 48 h.The number of eolonies resembling S, mutans on a predeter-mined area of the blade tip were estimated fbr each side of thetongue blade under a dissecting microscope. They were scoredon a seale of I to 5 with 1 = no growth, 2=1-10 eolonies,3=11-50 colonies, 4 = 51 100 eolonies, and 5 = more than 100colonies. Some de)iibtful eolonies were isolated on agar and testedfor biochemical and tnorphole)gie eharaeteristies,

MSBB The MSBB method is base d on the ability of S,mutans to adhere to glass surfaces when gre)wn in suerose-eon-

taining baeitracin broth (MSB bi-e)lh), 'This bi-e)lh is avaiUihlccommercially (Showa Yakuhin Go, Lid,, Tokyo, Ja]iaii) in ^sealed an-ipulc ihal permits stejrage without deterioration for 15me)ntlis, Bacitraein, trypan blue and tellurite are added to theMSB broth by elution from a dried filter strip immediatelybefore use, Unstimulated whole saliva was expectorated into asterile disposable eu|) anel 0,1 nil was wilhdrawn by means of asterile disposable pipette- and Iransfert-ed lo the ampule, whichwas held almost he)ri-/,e)ntally anel culturc-d aere)l)ie-ally al 37"C;for 24 h. The ampule was gently turned upside down three timesto remove any bacterial deposits adhering lejosely lo the glasssurfaee and seored on a modilied scale of 1 lo 5 using a referencepicture. In this modification of the original method (11) a scoreof + -t- + was lurlher divide-d into 4 or 5 according lo tbenumber of the adherent ce)le)nies,

Statistieal analy.sis and ettmparison of eost anil lime required T b erelation between caries increments (DMFS) for the previous 4yr and the seores obtained from Iwo methods was examined byregression analysis and cln-sc|uat-e lesls, 'The c-orrelalion betweenthe sc-ores ol Ihe twcj methods for eaeh individual subjeet wastested by ihrc-e statistical methods: regression analysis, ehi-squaretest and "overall kappa" test (14),

The eost of eaeh test, ineluding lhe time eae:h te-epiircd inpreparing iru:dium, sampling, eulluring, and sce)i-ing, was alscieompare:d.

RESULTSCowparison of S. mutans scores and DM PIS inerentent —

Distributions of the two S, mutans scores and DMFS

S,MUTANS SCORES

MSBA

MSBB

SCORE 1

DMFS INCREMENTS*

INCREMENTALGROUP 0-1 2-4 5-7 >8

-4-0 50

PERCENT100

* CARIES INCREMENTS FOR PREVIOUSFOUR YEARS

t''ig. I. PereenI dislribulion of S, mulaus seores by MSBA andMSBB methods and of earies inerements for previous 4 yr (brapproximately 300 ehildren in ihis sludy allending six publieschools, *Garies iueremenis for |)n-viocis 4 yr.

Screening tests for S. mutatis 327

T'able 1, Correlation between MSBA score and DMFS incrcmeiiKs" of individual children

MSBA score 0-1 iiicr,DM1*'8 increment gronps

2-4 incr, 5-8 incr, 8 or greater iner. Total

3620282414

15102320

54

131316

2036

15

5834676370

Total 122 93 292

Chi-square analysis: x'=41,12 (/'<0,0001),Regression analysis: r = 0,35 (P<0,01)," For previous 4 yr,'' Values are miniher of children in each group.

Xablc 2, Gorrelalion between MSBB ,scdrc and DMKS increments'' of individual children

MSBB scoreDMFS inerement groii])s''

0 1 incr, 2 'I incr, 5 8 incr, 8 or greater incr. Total

4113162426

228

171431

63

101120

22I7

13

7126445690

Total 120 92 287

Chi-square analysis: X' = 32,22 {P<O.OOi).Regression analysis: r = 0,26 (/'<0,01),^ For previous 4 yr,'' 'Values are number ofchildren in eaeh group.

increment group,s in the children examined are stmi-marized in Fig, 1, S, mtttan,s was detected in mostchildren (about 80%) by both methods (lower limitof detection is ^ 10 bacteria/ml ,stimnlatcd saliva forMSBA metbod and ~ 10'' bacteria/ml unstiniulatedsaliva for MSBB method). Almost half of the school-children had more than 10 S, mutans per ml, and3 0 % bad more than lO"*, However, the averagecaries increment was low; niatiy children (42"/n)developed one or no lesions (DMFS) during the 4yr. Only a lew children (9%) developed eight ormore lesions (DMFS) during the study.

Correlation between S. mutans scores and past DMFS

increments There were positive correlations ()' =+ 0.35 and +0,26) between DMFS increments forthe previous 4 yr and S, mutans scores as deter-mined by bolh Ihc MSBA and the MSBB tests(Tables 1 and 2), About 60% of the children withan MSBA score of 1 or 2 had a DMFS incrementof 0 to 1 duritig the previotis 4 yr. Conversely, alittle less tliati half of the children with ati MSBA

score of 5 had a caries increment of 5 DMFS ormore; most ofthe children (80%) with 8 or greaterDMFS increments had MSBA scores of 4 or 5,Similar results were obtained by the MSBB method.If children are classified according to DMF incre-ments of greater or less than 8, it is apparent (Table,3) that for an MSBB score of 5, 185 children (64%)were idctitified as true negative. Conversely therewere 77 children (27%) with MSBB scores of 5 whowere false positive. These tests successfully identifychildren with low caries increments, but positive

Table ,3, Classification of cliildreii with DMFS increments ofgreater or less than 8 b>' MSBB scores

MSBBscore

234,'j

Falseposilive

193169121)77

Falsenegative

24,••)

12

Truepositive

23212013

Truenegative

6993

13618,'')

328 NEWBRUN ET AL.

Table 4, Ratio scores for children by MSBB score and DMFS increments greater or less than 8

MSBBscore Sensitivity'' Specificity'

Value ol|)ositive test'

0,1 10,110,140,14

Value olnegative test

0,970,960,960,95

0,920,840,800,52

0,260,350,520,71

^ Sensitivity = 1-P, where (3 is Type II error (false negative),'' Speciticity= 1-a, where a is Type I error (false positive),'^ Value of positive test is ratio of true positive to all positive scores,• Value of negative test is ratio of true negative to all negative scores.

scores do not correlate well with higb caries inere-ments (Table 4), Children were grouped accordingto their S, mutans scores by both MSBA and MSBB,and the mean DMFS increments of each group werecalculated (Fig, 2 and Table 5), There were morestatistically significant differences in mean DMl' Sincrements bet'ween S, mutans scores by the MSBAmethod than by the MSBB method. However, byboth methods caries increments inereased witb in-creasing S, mutans score (except between S, mutansscores I and 2 by MSBA method, see Table 5),

Comparison of MSBA and MSBB S. mutans screeningtests - Tbe correlation between tbe two tests incategorizing tbe degree of S, mutans infection in

LU

LU ,—DC CO

LU CODC LJ.

< :EO Q<LU

MSBA—••

MSBB—A-

1

STREPTOCOCCUS MUTANS SCOREFig. 2. Correlation between mean caries increment (DMFS/4yr) an(J S, mutans seores by MSBA and MSBB methoiJs forapproximately 300 ehildren in this study attending six publicschools. Lines drawn by linear regression analysis.

each child was significant by three statistieal anal,-yses (Fable 4), No dillerenees or diflerenees of onlyone score were observed in 83% of paired cases.

Table 5, Mean DMFS increments'' in MSBA and MSBB .seoregroups

S, mutans seoreMean DMFS increments''

MSBA group'' MSBB group''

12345

1,76 + 2,471,62 + 2,072,69 + 2,493,14 + 2,784,49 + 3,34

(58)(34)(67)(63)(70)

1,83 + 22,,'jO ± 22,89 + 23,16 ±33,64 + 3

,45,69,41,14,07

(71)(26)(44)(56)(90)

''' For previous 4 yr,'' Values are means + SD (number of subjects).For MSBA, there were signifieant difl'erences (/-test) betweenscores 1 and 3 (/^<0,05), 2 and 3 {P<0.Q25), 1 and 4, 1 and 5(/'<O,OI), 2 and 4, 2 and 5, 3 and 5, 4 and 5 (/'<0,005),l"or MSBB there were signifieant dillerenees belween seores 1and 3 {P<0.025), I and 4 (/'<O,OI), 1 and ,') (/'<0,00,'j).

Table 6, Correlation of S, muians scores by MSBA and MSBBfor individual ehildren^

MSBB score

12345

Tolal

1

49

CT

)

223

62

MSBA2

109761

33

3

87

221910

66

score4

4-28

1831

63

5

33(i

1047

69

Total

7427455592

293

" Values represent individual children seored by both methodsStatistical analysis:

Chi ,square analysis: x ' = 214 (/'<0,001),Regression analysis: r = 0,68,"Kappa" statistical analysis: ,j= 7,047,

Screening tests for S. mutans 329

T'able 7, Compari.sdu of practical variables for both tests

'V;iriable,s

Xirne ]jer child''Sain|)lingCulturiugScoring

Cost per child''

MSBA lesl

2 min48 h''1 mill'$1,15

MSBB lesl

1 mill24 h

0,5 min$1,25

" 'Times for pi'e|)aralioii of both tests were not inchided,K(]iiipment for anaerobic culture is required,

• T"o identify eolonies grown on the agar as S, mutans, a dissect-ing microscope is i'('(]iiii'ed. Donblfhl coldnies re(]uire addi-tional biochemieal lests.Personnel expenses Ibr the preparation of MSB agar and seor-ing of both tests were ineluded, C osl of MSBB sup|)Iies formass screening estimated by manufacturer.

Several practical characteristics of the twomethods when used for mass screenings were com-puted (Table 7), Time for sampling, culturing andscoring ofthe MSBA method was twice that of tbeMSBB method. Both methods cost almost the same;cost estimates included the personnel expense forpreparation and ,scoring. In this study, si,\ doubtfulcolonies on MSB agar were observed and biochemi-cal tests were performed to confirm their identifica-t ion as S, mutans.

DISCUSSIONT h e pressing need for triage (selection) in the deliv-ery of prevetitive dentistry regitnens to those pa-tients who arc riiost in need of such intervention iswell recogni'/ed, A National Preventive DentistryDemonstration Program has been conducted re-

'- gionally iti 10 sites to determine costs and effective-: ness of combinations of school-based preventive pro-

cedures involving approximately 25 000 children in• grades 1 to 8, About 20'/o <5' l bc ebildren acquired

60 % the caries inerement (15), Similarly, in areeent U,S, National Detilal (Varies Prevalence Sur-vey (1979 80), 23,6",, ofthe children had 5 or moredecayed, tnissing or filled tcclh (DMFT), whereas3 9 . 8 % 1 to 4 DMFl^ and 36,6'^ were carics-free (16), In Denmark, Norway and Sweden,despite an overall decrease in dental earies in chil-d ren , a small fraction of the children still demon-strate relatively high caries prevalence (17-19), InS'witzcrland, after 12 yr ofa school-based caries-preventive regimen and demonstrable caries redue-tion, a small mitnbcr of the children had a higher

proportion of cavities, a phenomenon termed "pola-rization" by MARTHALER (20), If this high-needpopulation could be identified early, before the de-velopment of carious lesions, the cost/efficacy ratioof preventive treattnent would be more favorablebecause it could be directed to those ehildren atgreatest risk. In a recent analysis of the cost valueof a caries preventive program (supervised dailybrushing with a fluoride dentifrice and professionaltopical application of APF), the most favorable ratiowas obtained when only the high-risk group hadbeen treated (21), In this analysis the cost of pre-vention for both the general population and thehigh-risk group exceeded the estimated saving inrestorative treatment. However, this was based ondental costs in the British National Health Service;in private practice the restorative fees would begreater but the efficacy of prevention the same.Accordingly, the cost value would be more accept-able in countries with private dental services,

A good caries predictive test should possess atleast three characteristics: validity, reliability, andfeasibility (4), Validity implies predictive validity, sothat a child placed in a high caries activity (positive)category and followed for years should demonstratehigh caries increments, while a child in a low cariesactivity (negative) category should have minimalor no caries increments. In other words, thereshould be a minimum of false-positive or false-nega-tive test results, but this is difficult to achieve inpractice because no single test can simultaneouslymeasure host susceptibility or resistance, microbialpathogens, and cariogenicity ofthe diet (3),

Our results showed a positive correlation betweenS, mutans scores and past caries increments. Bothtests were very good for identifying caries-inactivechildren but were less satisfactory in identifying car-ies-active children, as some who had positive scoresdeveloped little caries. The reliability of both theMSBA and MSBB te,sts has been doctimented pre-viously (12),

Ideally, caries activity tests are performed at thebeginning of a clinical study and the results com-pared with subsequent caries increments. In thiscase S, mutans scores were compared to the cariesincrement of the preceding 4 yr. Although this ispreferable to a eomparison with caries prevalence,which represents cumulative caries experience overmany years, it must be recognized that S, mutanscounts and caries activity could change in an indivi-dual child. However, a study of 53 tmtreated ehil-

330 NEWBRUN ET AL.

dren, 13-14-yrold, found that most showed remark-able stability in tbe number of salivary S, mutansduring 3 yr of repeated counts (22), We would haveliked to further subdivide tbe caries incrementaldata to determine if there were better correlationswitb the incremental year immediately precedingtesting. Unfortunately, such annual incrementaldata were not available to us.

Although this study was conducted on a controlpopulation (one residing in a nonfluoridated com-munity and not receiving any specific carics-prcven-tive therapy), the overall caries inerement was rela-tively low. Less than 10% ofthe children experi-eneed more than 2 DMFS per yr (> 8 DMFS dur-ing 4 yr). This is in accord with other observationsof deelining earies prevalence in U,S, schoolchildren(23), Because of this low caries inerement, skewedto the right (Fig, 1), there were fewer children withhigh caries inerements, making it less likely that wewould obtain good eorrelations with the higher S,mutans scores.

Both methods are simple and inexpensive. Theadvantages ofthe MSBB method are that the brothcan be supplied commercially in a form that willnot deteriorate for 15 months, and that it can beperformed by semi-skilled personnel who are notrequired to differentiate S, mutans colonies by mor-phology or biochemical tests. However, this methodhas a disadvantage in that it cannot be used forvery young children who have great difficulty inproviding unstimulated whole saliva. The advan-tages of the MSBA method are that it correlatesmore highly with caries increment than the MSBBmethod and thus has higher validity in identifyinglow-risk populations, and that it can be applied tovery youtig children who must be identified early.The disadvantages of the MSBA method are thatthe agar is stable for only a week after j^reparation,and microbiological training is needed to dilferen-tiate S, mutans eolonies because a few other speciesof streptococci are occasionally resistant to bacitra-cin and also grow on the agar. Accordingly, al-though both tests can be used for mass screenings,the longer storage capacity of the brotb and theabsence of need for a microbiologist make the MSBBmethod preferable for use in private practice,whereas the MSBA method's slightly higher correla-tion with caries increments makes it ,somewhat bet-ter for mass screenings, where storage capacity ofthe agar would not be a problem and where theservices of a microbiologist would be cost-effective.

Aeknowtedgments This study was supported in part by the RobertWood Johnson Foundation, Caries ineremeiital data were |3ro-cessed by the Rand Cor]>oration under the direction of Ors. SP, Ki.EiN and R, M, BEi,t,, The authors are indebted to Ms, E,LioASM for editorial assistanee.

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