Suitability of throat culture procedures for detection of group A streptococci and as reference standards for evaluation of streptococcal antigen detection kits

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Copyright C 1990, American Society for Microbiology

MINIREVIEW

Suitability of

Throat

Culture Procedures

for Detection of Group A

Streptococci

and

as

Reference Standards for Evaluation of

Streptococcal

Antigen Detection Kits

JAMES A. KELLOGG

ClinicalMicrobiology Laboratory, York Hospital, York, Pennsylvania 17405

INTRODUCTION

There have beennumerousconflictingreportsconcerning the mostefficient culture methods for detection of groupA

streptococci. Significant variables in this procedure have

includedtheselection of inhibitoryversusnoninhibitoryagar

media and the atmosphere (aerobic, aerobic with 5 to 10% C02, oranaerobic)andduration (1dayversus2) of incuba-tion. This review documents studies that have addressed thesevariables,comparestheir methods andinterpretations,

and, in light of thesecomparisons, makesrecommendations for theselection of suitable throat culturereference methods

foroptimal detection ofgroup Astreptococci and for precise evaluations ofnewrapid antigen detection kits.

NEEDFOR AN ACCURATE CULTURE METHOD

Theproperevaluation ofany newdiagnostictestrequires

that the investigator be able to precisely determine the ability of a new procedure, compared with an accurate

reference test or "gold standard," to detect or exclude a

disease (38). The throatculture, however, servesmerelyas a screen for the presence or absence ofgroup A

strepto-cocci. If asensitive cultureprocedure results indetection of either fewormanycolonies oftheorganism,thepatientmay beinfectedor merelycolonized (13, 21, 23). Forclinicalas

wellas technicalreasons, there is no significantcorrelation between colony count and the presence or absence of infection (23). Differentiation of infection from colonization

requires the demonstration of an antibody response to the

organism,

aresponsewhichis bothtime-consuming (requir-ing 2 to 3 weeks or more between serum samples) and

subjecttofalse-negativeresults following prompt and

appro-priate antibiotic therapy (5, 14, 24, 49). If group A strepto-cocci are not detected from a symptomatic patient when a

sensitive throat culture technique is used, however, the chance that thepatient has been recently infected with the

organismshould be extremely small (9).

The clinical utility ofa good throat culture

procedure

is in

thehigh

predictive

valueof a negative result (PVN) and in thephysician's ability to avoid indiscriminate use of antibi-otics whenStreptococcus

pyogenes

can be confidently ruled outas a cause of

pharyngitis

(46). If a carefully chosen and

performedthroat culture method has a sensitivity of 95% and aspecificityof99%(asdocumented by comparison with one or more of those

procedures

listed in the Conclusions

section), the PVN will be excellent in low- or

moderate-prevalence populations (99.0 and 97.9% with moderate-prevalences of 15 and 30%, respectively) but will drop steadily when the prevalence rises (96.1% with a prevalence of 45%, 93.0%

with a prevalence of60%, and 86.7% witha prevalence of

75%).

If the throat culture is

poorly performed

and has a sensitivity of only 75% with a specificity of 90%, the PVN

mightbedeemedacceptablein alow-prevalence population (95.3% withaprevalenceof15%)but isclearlyunacceptable inahigher-prevalence population

(81.5%

witha prevalence of

45%).

Becauseatestwithasensitivity of only 75%misses oneinfectedpatientoffour, itshould either beimprovedor

replaced, regardless ofthe prevalence of infection, when-everthere aresuitable alternatives.

A newstreptococcalscreenprocedure (cultureorantigen assay) shouldhave asensitivity ofatleast 90 to 95% when

compared to a competent reference culture (as listed in

Conclusions)

so that almost all infected

patients

will be detected(and specifically treated),the PVN will be

accept-ablyhigh, and antibiotics can be withheld ordiscontinued withconfidencefrom uninfected patients. Themore accurate thereferencemethod, themorereliable will bethe resultsof

comparisons performed witha newalternativetest(38).The

reported sensitivity of the directtests for group A

strepto-cocci has been lower when the reference throat culture

technique included a selective medium or the culture was incubated fora second day or under anaerobic conditions (40). Interpretation of true kit performance

(sensitivity,

specificity, and predictive values ofresults) can be made

with confidence only if investigators use a throat culture techniquewithprovenperformanceas the "goldstandard"

and

provide

details of that

performance

(23)from theirown

investigations or by referring to the published studies of others. An

analysis,

basedon areview of24

investigations

(Table 1), of three of the more significant and controllable variablesinthroat culturemethods follows.

VARIABLESINTHROAT CULTURE TECHNOLOGY

Duration of incubation. While there have been many

conflicting data on other aspects of throat culture

proce-dures,almost all authors have agreedthatcultures negative for group A streptococci following overnight incubation should be incubated for another24h. Increasedrecoveries

of S. pyogenes when 5% sheep blood agar (SBA) cultures wereincubated for a second day ranged from 5 to46%in five studies (Table 1) using aerobic incubation (17, 29, 31, 36; J. A. Kellogg and D. A. Bankert, Abstr. Annu. Meet. Am. Soc. Microbiol.1989,C123, p.414), 9 to35%in twostudies

with

C02-supplemented

aerobic incubation(31,36), and 2 to

31%in sevenstudiesusing anaerobic incubation(17, 22, 29, 31, 35, 36; Kellogg and Bankert, Abstr. Annu. Meet. Am. Soc. Microbiol. 1989). A second day of incubation of SBA

containing sulfamethoxazole and trimethoprim (SBA-SXT) 165

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TABLE 1. Recoveryofgroup A streptococci by using differentmedium-atmospherecombinations

No.(%) of group A streptococci recoveredfromthefollowingmedium-atmosphere combinations:

Refer- Totalno.of Duration of SBA SBA-SXT ssA

ence positive incubation

cultures (h) Aerobic Aerobic Aerobic

Aerobic (5-10% Anaerobic Aerobic (5-10% Anaerobic Aerobic (5-10%

C02)

C02)

C02)

3lb 24 294(16.3) 209(11.6) 236 (13.1) 191 (10.6) 211(11.7) 224(12.4) 48 327(18.1) 282(15.6) 309(17.1) 312(17.3) 321(17.8) 309(17.1)

36C 24 56(21.2) 68(19.5) 60 (23.5)

48 59(22.3) 74(21.2) 61(23.9)

17 37 24 24(65) 29(78)

48 35(95) 37(100)

41 201 36-48 180(89.6) 195 (97.0) 167(83.1)

29 200 24 138(69.0) 182(91.0)

48 174(87.0) 193(96.5)

_d 258 24 213 (82.6) 233 (90.3)

48 254(98.4) 255(98.8)

1se 18-24 50(86.2) 73(85.9) 134(93.7) 142(99.3)

3 48 24 14(29.2) 48(100)

2 1,041 18-24 843(81.0) 1,036 (99.5)

39 218 18-24 178(81.6) 207 (95.0)

îîf 18-24 94(63.1) 146 (98.0) 57 (70.4) 112(83.6)

45 1,479 18-24 1,054 (71.3) 1,467(99.2)

1 58 18-24 45(77.6) 44(75.9)

34 31 18-24 16(51.6) 31(100)

35 201 24 159 (79.1) 114(56.7) 155(77.1)

48 170(84.6) 185(92.0) 193(96.0)

27 102 24 93(91.2) 102(100.0)

48 78(76.5) 91(89.2) 100(98.0) 102(100.0)

379 18-24 64(8.8) 72(9.9)

26 204 18-24 157(77.0) 162 (79.4) 179(87.7) 185(90.7)

22 163 24 154(94.5) 134(82.2)

48 161(98.8) 158(96.9)

7 265 24 244(92.1)

48 243(91.7) 262(98.9)

19 243 48 233(95.9) 225(92.6)

16 55 18-24 31(56.4) 54(98.2)

48 50 18-24 36(72) 47(94)

h 138 48 126(91.3) 125(90.6)

a Except where indicated, results show percentages of total numbers of isolates of groupAstreptococci.

b Results show percentages of 1,805specimens which contained groupA

streptococci.

cResults show percentages of 264aerobic,349aerobic(CO2), and 255 anaerobic SBA cultures which contained group A

streptococci.

dKellogg and Bankert, Abstr. Annu. Meet. Am. Soc. Microbiol. 1989.

e Results show percentages of58

aerobic

SBA, 85 aerobic (CO2) SBA, 143 anaerobicSBA, and143aerobic(CO2) ssA isolates of group A

streptococci.

fResultsshow percentages of 149 aerobicSBA, 149 anaerobic SBA, 81 aerobicSBA-SXT,and 134 anaerobic SBA-SXTisolatesof group Astreptococci.

g Results show percentages of 725 specimens which contained group Astreptococci.

h T. A. Kurzynski, M. B. Polyak, C. J.Sprecher,R. F.Schell,Letter, Eur. J. Clin. Microbiol.

6:602-603,

1989.

resulted in increasedrecoveriesof theorganism of 62 to 63% in two studies with aerobicincubation (31, 35), 8 to52% in

threestudies withC02-supplemented aerobic incubation(22, 27, 31), and 0 to 38% in three studies using anaerobic incubation(27, 31, 35). Of23 medium-atmosphere combina-tions (from nine studies; Table 1) for which recoveries at1 and 2 days of incubation were compared, recovery in 22

(96%)wasimprovedonthe seconddayand theincrease was morethan5%in20(87%).Toachievemaximumdetectionof

group A streptococci, a high PVN, and a high degree of confidence inresultsfrom studies which include streptococ-cal cultures, those cultures negative at 1 day should be incubated for a second day, regardless ofmedium or atmo-sphere selected, unless a thorough in-house investigation indicates that a second day of incubation has been docu-mented to be unproductive. Delayed detection ofgroup A streptococci is due to slow growth or lack ofhemolysisat24 hofsome strains (44).

Inhibitory versus noninhibitory media. Group A

strepto-cocci present in a specimen may be missed in culture

because of factors that include the ability oforopharyngeal

normal flora (e.g., Staphylococcus species, Neisseria spe-cies, andviridansgroupstreptococci)toovergrowthe

patho-gen (7, 26, 27, 31, 39) and mask its expression of beta-hemolysis (3, 7, 34). In addition, other bacterial species

encountered in the throat have been shown to inhibit the growth of S. pyogenes by production of either viridins (bacteriocins produced by viridans group streptococci) or other chemicals (such as hydrogen peroxide or acids) toxic tobacterialmetabolism,aswellasby depletion ofsubstrates (4, 10, 18, 30, 42, 43, 47; W. E. Sanders and C. C. Sanders, Clin. Microbiol. Newsl. 4:127-130, 1982).

Inclusion of SXT in SBA has been shown to selectively

inhibit many of the normal floralspecies (16) byblockingthe pathway for microbial productionoffolic acid(6, 25). Some

organisms, such as group A and B streptococci and Entero-coccus species, can utilize exogenous thymidine in the medium to circumvent the pathwayinhibited by SXT. Oth-ers, including other groups ofbeta-hemolytic streptococci,

viridans group streptococci, and Streptococcus

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niae, cannot, eveninthepresenceofhigh concentrations of thymidine, and remain inhibited (8, 25). Because Trypticase

soy agar is a natural infusion medium, there is much

lot-to-lot variation in its concentration ofthymidine and thusin theability of different lots of SBA-SXT (made from

Trypti-case soy agar) to suppress the growth of some species of

normalflora (1, 8, 11, 27). A semisyntheticagarbase witha

defined, constantconcentration of thymidine would be less likelytoresult in significant variations in growth ofsomeof

the normal floral species with different batches of the

me-dium (8).

Ofeight studies comparing rates of detection of S.

pyo-genes on SBA and SBA-SXT, five found that the recovery

wassignificantly improvedontheselective medium(Table 1; 16, 26, 27, 35, 48), but cultures were incubated for 48 h in

only two of these studies (27, 35). The only investigation which compared recoveries ofgroupAstreptococcionSBA

incubated inall three atmospheres (aerobic;aerobic with 5 to 10% C02, anaerobic) with recovery on SBA-SXT similarly incubatedreported that detection of the organisms wasbest (and not significantly different) either on SBA incubated

aerobicallyor on SBA-SXTincubatedaerobicallywithCO2,

both for 48 h (31). Only oneof the eight studies foundthat

SBA(incubated anaerobically) provided significantly greater detection of S.pyogenesthan didSBA-SXT,but the cultures

wereheld forjust 18to24 h (11). Variationsin reports of the

recovery of group A streptococci on SBA-SXT compared

with recovery onnoninhibitory SBA are most likelydue to

factors that includevariations in thethymidinecontentof the different media used(8, 11, 27), the differentsusceptibilities ofsome normal floral speciesto SXT (8), variations among

different populations in the species making up the normal flora (35), and significantly improved recovery of S.

pyo-genes when SBA-SXT cultures are incubated for 48 h (22, 27, 31, 35). Ail four investigations comparingnoninhibitory

SBA with SBA-SXT and in which cultures were incubated for 48 h found that recovery of group A streptococci on

SBA-SXT was either equivalent or superior to that on the best of theSBA-atmospherecombinations(22, 27, 31, 35).In addition, SBA-SXT cultures incubated eitheraerobically (5

to 10% C02) or anaerobically were shown to result in detectionratesof 96.9% (22)to98.0%(27)and 96.0%(35)to

100% (27), respectively, ofthe total groupA streptococcal isolatesrecovered, while anaerobically incubated SBA

cul-turesledtorecoveryof 84.6to98.8% ofthetotalisolates(22,

27, 35).

Theperformance of SBA containing colistin, crystal vio-let, and SXT (ssA)maybe similartothat ofSBA-SXT(Table 1), but more studies are needed. Of four evaluations

com-paringssA cultures with noninhibitory SBAcultures, three reported that detection ofgroup A streptococci was either

equivalentorsuperioronssA. However,inone(15) ofthese three, cultureswereincubated foronly 18to24 h and in the othertwo(7, 19), only SBA incubated inanaerobic(5to10%

CO2) environment was used for the comparison. As dis-cussed in the next section, SBA so incubated is likely to resultinarelatively lowrecoveryof S.pyogenes.The fourth study (41) reported that SBA cultures incubated either aerobically (without C02 supplementation)oranaerobically

resulted inrecoveryofsignificantlymoreisolates ofgroupA streptococci than didaerobically incubated ssA cultures.

Atmosphere of incubation. Therearethreepotential

advan-tagesofincubating cultures for recovery ofgroupA

strep-tococciinananaerobic atmosphere: detection of hemolysis

by strains of the organism abletoproduce only oxygen-labile streptolysin O(3, 11, 12, 17, 34, 36) and prevention of both

overgrowth

and

growth

antagonism by

other

(normal floral)

species

which

frequently

grow

poorly

(3, 11, 17, 27)

or

possessdiminished antibacterial

activity

(18)

inan

oxygen-free environment.

Of

seven

investigations

comparing

the

performances

ofSBA cultures incubated

anaerobically

with thoseincubated

aerobically

and/or

aerobically

with 5to 10%

C02

andin which cultureswereheldfor48 h, six

reported

that the recovery ofS.

pyogenes

on the anaerobic medium was either

equivalent

or

superior

to that on the aerobic

medium,

withorwithout

C02

(17,

27, 29, 36, 41;

Kellogg

and

Bankert,

Abstr. Annu. Meet.

Am.

Soc.Microbiol.

1989).

As

canbeseeninTable

1,

fiveof these six studies

reported

their total numbers of group A

streptococcal

isolates and four observed thatatleast

96.5%

oftheseisolateswererecovered

within48 hon

anaerobically

incubatedSBA. Infourstudies

comparing 48-h,

anaerobically

incubated SBA cultures with culturesgrownon

SBA-SXT,

recoveries ofS.

pyogenes

on

the mediawere

equivalent

in two ofthe

investigations

(22,

31)

but

superior

onSBA-SXTincubated either

aerobically

(5

to10%

C02)

(27)

or

anaerobically

(27, 35)

in the othertwo.

In the York

Hospital laboratory,

the cost associated with

both laborand theuse of

gas-generating envelopes

tocreate an anaerobic environment is

approximately

$0.13

per cul-ture.

Aerobicincubation of SBA

cultures,

like anaerobic

incu-bation,

results in less

overgrowth

ofS.

pyogenes

by

species

of thenormal

oropharyngeal flora

thandoesincubation inan

aerobic,

C02-supplemented atmosphere

(31).

Insix

investi-gations

comparing

recoveriesonSBAcultures incubated for

48 h both

aerobically

(without

C02)

and

anaerobically,

aerobic incubation resulted in

detection

ofS.

pyogenes

that

was

superior

in one

study (31),

equivalent

in three

(17, 36;

Kellogg

and

Bankert,

Abstr. Annu. Meet.

Am.

Soc. Micro-biol.

1989),

and inferior in two

(29, 41).

Variations in recovery of the

organism

on

aerobically

incubated SBA

among thesesix studiesmay be

due,

in

part,

tomethodsused toreducethe oxygentension inor over

part

ofthesurface of

the

aerobically

incubated culturesto detect

hemolysis

from

streptolysin

O.

Stabbing

the

medium,

as was done in four

studies

(29, 31, 36,

41),

mayworkwellif

carefully

performed

butcan

quickly

leadto gaps or

drying along

the stab lines,

resulting

inafailureto

sufficiently

reduce theoxygentension

(23, 32).

The use of a 22-mm2 cover

glass

placed

onto the

primary

inoculumzoneof each SBA culture

reliably

reduced

the oxygen tension and increased recovery of group A

streptococci

from SBA incubated

aerobically (in

10%

C02)

by

9.5%

(32).

Adistinct

advantage

of

aerobically

incubated SBA is that it results in a reduction in recovery of

beta-hemolytic

streptococci

other than group A

streptococci

when

compared

with recovery on SBA incubated either

aerobically

with

C02

supplementation (31, 36)

or

anaerobi-cally (11, 17, 22, 29, 31, 34, 36, 45).

This may result in a

significant savings

incosts associated with the

subculturing

or

serological

identification of these

streptococci.

The use of SBA incubated for 48 h in an aerobic

atmo-sphere supplemented

with 5 to 10%

C02

was found to

provide

either lower

(7,

27,

31)

or

equivalent (19, 36)

recovery of groupA

streptococci

when

compared

with other

medium-atmosphere

combinations. Because thenormal

aer-obic and

facultatively

anaerobic

species

grow sowellin an

aerobic,

C02-supplemented

atmosphere (31),

SBA cultures

soincubatedaremost

likely

toresult in the lowestrecovery of S.

pyogenes.

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CONCLUSIONS

No single medium-atmosphere combination is likely to

provide 100% recovery ofgroup A streptococci (37, 44).

Sampling variation alonemayleadtoafalse-negativerateof about 10% (21). However, from the results of carefully performed studies, each of the following medium-atmo-sphere combinations is likely, ifmeticulously processed, to

reliablydetectatleast 90to95%of thegroupAstreptococci

fromsymptomaticpatients: (i)SBAincubatedanaerobically for48 h (17, 22, 29, 36, 41); (ii) SBA incubated aerobically (withoutCO2supplementation) for 48 h (17, 31, 36, 41),using

a cover glass pressed onto the primary inoculum zone to

reduce oxygen tension (32); (iii) SBA-SXT incubated

aero-bically (5 to 10% C02) for 48 h (22, 27, 31); and (iv) SBA-SXT incubated anaerobically for 48 h (27, 35). As discussed above, SBA cultures in an aerobic,

C02-supple-mented atmosphere are likely to result in a relatively low recoveryofS. pyogenes. Therelative value of SBA-SXTin an aerobic atmosphere or ssAin anyatmosphere shouldbe

documented in further studies. Other medium-atmosphere combinations arenotdiscussed in thisrevieweitherbecause there arefew publications on theirperformanceor because

they are not widely used.

Several technical points should be stressed to maximize

recovery of S. pyogenes, regardless of the

medium-atmo-sphere combination which maybe selected.

(i) The specimen should beverymeticulouslycollected(as

previously described [23])to maximize the numberofgroup

A streptococci on theswab.

(ii) The swab should be firmly rolled over one-sixth (no

more) of the agar surface (12). This will ensure both that

organismspresentononlyonepartof the swab makecontact with the medium and that the medium isnotoverinoculated. (iii) The inoculum must be very carefully and efficiently

streakedacrossthe surface ofthemedium, inamannersuch

asthatdescribed byKaplan(20),tominimize overgrowth or

antagonismofS.pyogenesbyotherspecies andtomaximize

the ability to detect beta-hemolysis (34).

(iv) The culturesmustbe carefullyinspectedat 18to24 h and again (if negative at 1 day) at 48 h. The individuals reading the cultures shouldbethoroughly trained and

super-vised (23).

(v) Quality control of themedium,incubator,andreagents

used forrecovery andidentification of S.pyogenesis essen-tial (23). False-negative reports of group A streptococci

could beduetoproblems thatincludecertain lots of SBAon which beta-hemolysis cannot be demonstrated (34) and an

excessively high temperature ofincubation (28).

Many authors ofstudies onperformance of antibody kits

for direct detection ofgroupAstreptococci have related the

sensitivity of these products to the numbers ofCFU of the organismrecovered in culture. Aselective medium, suchas

SBA-SXT (16), or a selective atmosphere (aerobic [without

C02]oranaerobic) maymorereliablyindicate the number of

organisms onthe specimen byreducing the effects of normal

floralspecies. Whatevermedium-atmosphere combination is chosen for such astudy, the accuracyof its performance as

areferencemethodshouldbedocumented eitherby in-house

comparisons orbyreferring to published findings (or both), and cultures negative for S. pyogenes at 1 day should be incubated for a second day. Because of numerous recent

reports of the reemergence of rheumatic fever and the

appearance ofa toxic shock-like syndrome due to group A streptococci, "... it is vitally important to make every

effort toidentify allstreptococcalthroat infections and treat themadequately" (33).

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J.

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Clin. Microbiol. 27:1715-1718.

20. Kaplan, E. 1973. The throat culture: its techniques, pitfalls, limitations andmeaning. Conn. Med. 37:45-48.

21. Kaplan, E. L., F. H. Top, Jr., B. A. Dudding, and L. W. Wannamaker. 1971. Diagnosis of streptococcal pharyngitis: differentiation of active infection from the carrier state in the symptomatic child. J. Infect.Dis. 123:490-501.

22. Kellogg, J. A., D. A. Bankert, and J. S. Levisky. 1986. Suitability ofathroat culture method forevaluation of group A streptococ-calantigendetection kits. Am. J. Clin. Pathol. 86:624-628. 23. Kellogg, J. A., and J. P.Manzella. 1986. Detection of group A

streptococci in the laboratory of physician'soffice. Culture vs antibody methods. J. Am. Med. Assoc.255:2638-2642. 24. Kilbourne, E. D., and J. P. Loge. 1948. The comparative effects

ofcontinuous and intermittent penicillin therapy on the forma-tion of antistreptolysin in hemolytic streptococcalpharyngitis. J. Clin. Invest. 27:418-424.

25. Koch, A. E., and J. J. Burchall. 1971. Reversal of the antimi-crobialactivity of trimethoprim by thymidine in commercially prepared media. Apple. Microbiol. 22:812-817.

26. Kurzynski, T. A., and C. K. Meise. 1979. Evaluation of sul-famethoxazole-trimethoprim blood agar plates for recoveryof groupAstreptococci from throat cultures. J. Clin. Microbiol. 9:189-193.

27. Kurzynski, T. A., and C. M. Van Holten. 1981. Evaluation of techniques for isolation of group A streptococci from throat cultures. J. Clin. Microbiol. 13:891-894.

28. Larsson, P., and L. Lind. 1983. The need for controlofthroat streptococcalculturesin generalpractice. Scand. J. Infect. Dis. Supply. 39:79-82.

29. Lauer, B. A., L. B. Reller, and S. Mirrett. 1983. Effect of

atmosphereandduration of incubationonprimary isolation of groupA streptococci from throat cultures. J. Clin. Microbiol. 17:338-340.

30. LeBien,T.W.,andM. C. Bromel.1975. Antibacterialproperties ofaperoxidogenic strain of Streptococcusmitior(mitis). Can.J. Microbiol. 21:101-103.

31. Libertin, C. R., A. D. Wold, andJ. A. Washington H. 1983.

Effects oftrimethoprim-sulfamethoxazole and incubation atmo-sphereonisolation of groupAstreptococci. J.Clin. Microbiol. 18:680-682.

32. Marraro, R. V.1974. Reduced oxygen tensionfor recovery of (group A)streptococciin throatcultures. Am.J.Med. Technol. 40:41-44.

33. Massell, B. F., C. C. Chute, A. M. Walker, and G. S. Kurland.

1988. Penicillin and the marked decrease in morbidity and

mortality from rheumaticfever in the United States. N.Engl.J. Med. 318:280-286.

34. McGonagle,L. E.1974.Evaluation of a screeningprocedure for the isolation of beta-hemolytic streptococci. Health Lab. Sci. 11:61-64.

35. Mirrett,S., J.S.Monahan,and L. B.Reller.1987.Comparative evaluation of medium and atmosphere of incubation for

isola-tion ofStreptococcus pyogenes. Diagn. Microbiol. Infect. Dis. 6:217-221.

36. Murray, P. R., A. D. Wold, C. A. Schreck, and J. A. Washington Il. 1976. Effects ofselective media and atmosphere of incuba-tion on the isolaincuba-tion of group Astreptococci. J. Clin. Microbiol. 4:54-56.

37. Pien, F. D., C. L. Ow, N. S. Isaacson, N. T. Goto, and R. C. Rudoy. 1979. Evaluation of anaerobic incubation for recovery of group A streptococci from throat cultures. J. Clin. Microbiol. 10:392-393.

38. Radetsky, M., and J. A. Todd. 1984.Criteria for the evaluation

of newdiagnostictests.Pediatr. Infect. Dis.3:461-466.

39. Randolph, M. F., J. J. Redys, and J. B. Cope. 1984. Evaluation of aerobicandanaerobic methods forrecoveryofstreptococci from throat cultures. J. Pediatr. 104:897-899.

40. Reichwein, B., D. Jungkind, M. Guardiani, R. Gilbert, G.

Prosswimmer, and P. Amadio. 1986. Comparison oftwo rapid latex agglutination methods fordetection of group A streptococ-calpharyngitis. Am. J. Clin. Pathol. 86:529-532.

41. Roddey, 0. F., H. W. Clegg, L. T. Clardy, E. S. Martin, and

R.L.Swetenburg.1986.Comparison ofalatexagglutinationtest and four culture methods foridentification of group A strepto-cocciinapediatric office laboratory. J. Pediatr. 108:347-351.

42. Sanders, C. C., W. E. Sanders, Jr., and D. J. Harrowe. 1976.

Bacterialinterference: effects of oral antibioticsonthenormal throatflora anditsabilitytointerfere with groupAstreptococci. Infect. Immun. 13:808-812.

43. Sanders, E. 1969. Bacterial interference. I. Its occurrence amongtherespiratorytractflora andcharacterization of inhibi-tionof groupAstreptococci by viridansstreptococci. J. Infect. Dis. 120:698-707.

44. Schaub, I. G., I.Mazeika,R.Lee,M. T.Dunn,R.-A.LaChaine,

and W. H. Price. 1957.Ecologicstudies ofrheumatic fever and

rheumatic heart disease. I. Procedure for isolatingbeta hemo-lyticstreptococci. Am. J. Hyg. 67:46-56.

45. Schwartz,R.H.,M. A.Gerber,and P.McCoy. 1985. Effect of atmosphere of incubation onthe isolation of groupA strepto-cocci fromthroatcultures.J. Lab.Clin. Med. 106:88-92. 46. Stollerman,G. H.1982.Globalchanges in groupA

streptococ-caldiseases and strategies for theirprevention. Adv. Intern. Med. 27:373-406.

47. Tagg, J. R., A. S. Dajani, and L. W. Wannamaker. 1976. Bacteriocins ofgram-positivebacteria. Bacteriol.Rev. 40:722-756.

48. Tolliver,P. R.,M. H.Roe,andJ. K. Todd.1987. Detectionof group Astreptococcus: comparisonofsolid and liquidculture media with and without selective antibiotics. Pediatr. Infect. Dis. J. 6:515-519.

49. Wannamaker, L. W., C. H. Rammelkamp, Jr., F. W. Denny, W.R.Brink,H. B.Houser,E.0.Hahn,andJ.H.Dingle. 1951. Prophylaxis of acute rheumatic fever by treatment of the precedingstreptococcal infection with variousamountsofdepot penicillin.Am. J. Med. 10:673-695.

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Quantitation

of Group A

Beta-Hemolytic Streptococci

in Throat

Cultures

In Kellogg's review (7) of throatculture procedures for the recovery ofgroup A beta-hemolytic streptococci (GABHS), he rathersummarily dispenses with quantitation of GABHS on the culture plates as providing any indication as to the presence orabsence of true streptococcal infection. He cites twostudiesother than his own review article (8) as evidence for this statement. In the first, by Gerber et al. (4), there was no significantcorrelation between the degree of positivity of

throatcultures and changes in antibody titers. In the second,

by Kaplan et al. (6), although the correlation was not

significant, there was a trend toward the association of an

initialcultureof 2+ or greater (10 to 50 CFU of GABHS) and

serologic evidence of infection. Also, in a later study (5), Kaplan et al. found that there was a definite trend for

individualswith strongly positive cultures to demonstrate an

antibody rise. Similarly, in 127 streptococcal infections,

Miller et al. (10) found 45% of "grade 3" cultures to be

associatedwith at least a two-tube increase in

anti-strepto-lysin 0, as opposed to only 19 and 6%, respectively, for

"grade 2" and "grade 1" plates. They interpreted this as

showing a "clear-cut relationship between the number of

coloniesandthefrequency withwhicha significantincrease inantistreptolysin-O was observed."

Whereas the above-mentioned studies lead to differing conclusions on the basis of antibody data, those which

correlate clinical findingswiththroat culture results are very much in accord. In a private pediatric practice, Stillerman and Bernstein (11) compared positive cultures of 98

symp-tomaticand 96 asymptomatic cases and found that strongly

positive cultures were much more likely to occur in the

former group. Inculturing 1,054 children with sore throats

and462 whowere asymptomatic, Bell and Smith (1) found that 71% of the 350 positive cultures in the former group

showedaheavy growth ofGABHSas opposed to only 10%

ofthe 80positive isolates in the latter one. Thefindings of

Margileth and Mella (9) were similarwhen they compared children with pharyngitis and their asymptomatic siblings. Wannamaker(12) andBreese et al.(3)inreview articles and

a panel of experts assembled by the chiefeditors of the

Pediatric Infectious Disease Journal (2) all attest to the

clinical importance of the degree of

positivity

of throat

cultures for GABHS.

The vastmajority of studieswhichcorrelatepatient

symp-tomatology with throat culture

quantitation

support the

belief that quantitation is at least of some

importance

in

differentiating the streptococcal carrierstatefromtrue infec-tion. To ignorethese studiesmay be

misleading, especially

to those whoare

primarily

or

exclusively

in the

laboratory

and do not have the

opportunity

of

frequently

correlating

clinicaland

laboratory

results.

REFERENCES

1. Bell, S. M., and D. D. Smith. 1976. Quantitative throat-swab

culture inthediagnosisofstreptococcalpharyngitisin children.

Lancetii:61-63.

2. Breese, B. B.,F. W.Denny, H.C. Dillon, M.Stillerman, J.D.

Nelson,and G. H.McCracken,Jr. 1985. Difficultmanagement problems in children with streptococcal pharyngitis. Pediatr.

Infect.Dis. 4:10-13.

3. Breese,B.B.,F. A.Disney,W.B.Talpey,andJ.L.Green.1970. Beta-hemolyticstreptococcal infection. Am. J.Dis. Child. 119: 18-26.

4. Gerber, M. A., M. F. Randolph, J. Chanatry, L. L. Wright.

K.K.DeMeo, and L. R. Anderson. 1986. Antigen detectiontest

for streptococcal pharyngitis: evaluation of sensitivity with

respectto true infections. J. Pediatr. 108:654-658.

5. Kaplan, E. L., R. Couser, B. B. Huwe, C. McKay, and L. W.

Wannamaker. 1979. Significance of quantitative salivary

cul-turesforgroup A andnon-group A B-hemolytic streptococci in

patients with pharyngitisand in their family contacts.Pediatrics 64:904-912.

6. Kaplan, E. L., F. H. Top, Jr., B. A. Dudding, and L. W.

Wannamaker. 1971. Diagnosis of streptococcal pharyngitis:

differentiation of activeinfection from the carrier state in the

symptomaticchild. J. Infect. Dis. 123:490-501.

7. Kellogg, J. A. 1990. Suitability of throat culture proceduresfor

detection ofgroup Astreptococciand as referencestandards for evaluation of streptococcal antigen detection kits. J. Clin. Microbiol. 28:165-169.

8. Kellogg, J. A., and J. P. Manzella. 1986. Detection of groupA

streptococci inthe laboratory or physician's office. Culture vs antibody methods. JAMA255:2638-2642.

9. Margileth, A. M., and G. W. Mella. 1966. Office diagnosis of

respiratory-tract bacterial infections. Med. Ann. D.C. 35:245-249.

10. Miller, J. M., S. L. Stancer, and B. F. Massell. 1958. A

controlled study of beta hemolytic streptococcal infection in rheumaticfamilies. Am.J. Med.25:825-844.

11. Stillerman,M., and S. H. Bernstein. 1961. Streptococcal phar-yngitis.Am.J. Dis.Child. 101:476-489.

12. Wannamaker, L. W. 1965. Amethodforculturingbeta hemo-lytic streptococcifrom thethroat.Circulation32:1054-1058.

0. F.Roddey,Jr.

2711RandolphRoad, Suite 501 Charlotte, North Carolina 28207 Author'sReply

Iappreciate Dr. Roddey'scommentsconcerning the ques-tion of correlaques-tion of numbers of colonies of group A streptococci recovered on throat cultures with confirmed streptococcal infection. This kind ofdialogue ishelpfuland necessary bothtoclinicalmicrobiologists, such asmyself,in understanding the relevance ofthe tests which we perform andtoclinicians, in understandingthe strengths and limita-tions of thesediagnostic assays.

A subcommittee (composed primarily ofphysicians with extensive backgrounds in the diagnosis of streptococcal infections) oftheAmericanHeart Association has concluded that ". . . culture does not reliably distinguish between acute streptococcal infections and streptococcal carriers with concomitant viral infections" (4). They further state that "sparse growth of group A streptococci does not necessarily reflect the carrier state and may indicate acute infection." These conclusions, shared by numerous other clinicians (1, 3, 5, 7, 8, 12-14, 16),werepreciselythepoints ofthe comments, referred to by Dr. Roddey, which ap-peared in the review of group A streptococcal culture procedures (9). The throat culture is not designed as a quantitatively precise diagnostic test, and its results should not be arbitrarily interpreted as if it were. While many patientswhose cultures containedlargenumbers ofgroupA streptococcalcolonies have been shown to have asignificant

cultures contained either small numbers of colonies of the organism (5, 8, 12-14) orno suchcolonies atall (7, 12-14).

There aremany clinical and methodological explanations

for the lack ofcorrelation between numbers of streptococcal colonies inculture andanantibody response. Theseinclude antibiotic pretreatment ofpatients, duration of symptoms, variation in thoroughness of specimen collection, and the medium, atmosphere, and duration of incubation selected for culturedetection of the pathogen,aspreviously reviewed

in detail (9, 11). Dr. Roddey's pediatricgrouphas reported

that, depending on the type of culture medium and the atmosphere ofincubation tested, one type ofagarmedium

missed from 3 to17% of culture-positive patients (15). Ina

study that the York Hospital laboratory undertook with a

local pediatric group, the semiquantitative recoveries of

group A streptococci were the same from duplicate swabs

(onecultured in the pediatric office, the other in the labora-tory)obtained from only 60% of theculture-positive patients (10).

It appears clinically unwise to arbitrarily interpret a

cul-turecontaining onlyafewgroupAstreptococcalcoloniesas

coming fromacarrier rather thananacutelyinfected patient.

Similarly, differentiation of the carrier from the truly in-fected patient cannot always be reliably made on clinical

groundsalone (3, 4, 8)oreven,perhaps, from theabsence of an antibody response in a culture-positive patient (6). The

clinicianshoulduseallavailable clinical,bacteriological, and

epidemiological information together to differentiate a

pa-tient whomaybeacarrier fromonewithanacuteinfection (2, 16).

REFERENCES

1. Bisno, A. L. 1977. Therapeutic strategies for the prevention of

rheumatic fever. Ann. Intern. Med. 36:494-496.

2. Breese, B.B., F. W. Denny, H. C. Dillon, M. Stillerman, J. D.

Nelson, and G. H. McCracken, Jr. 1985. Consensus: difficult

managementproblems inchildren with streptococcal

pharyngi-tis. Pediatr. Infect. Dis.4:10-13.

3. Cornfeld,D., and J. P. Hubbard. 1961. Afour-year study of the

occurrenceofbeta-hemolyticstreptococci in64school children. N. Engl.J. Med. 264:211-215.

4. Dajani, A.S., A.L. Bisno,K. J. Chung, D. T. Durack, M.A. Gerber, E.L. Kaplan, H. D. Millard, M. F. Randolph, S.T. Shulman, and C. Watanakunakorn. 1988. Prevention of

rheu-maticfever. Astatementfor health professionalsby the

Com-mitteeonRheumatic Fever, Endocarditis, and Kawasaki

Dis-easeofthe CouncilonCardiovascularDisease in the Young, the

AmericanHeart Association. Circulation 78:1082-1086. 5. Gerber, M. A., M. F. Randolph, J. Chanatry, L. L. Wright,

K.K.DeMeo,and L.R.Anderson. 1986.Antigen detectiontest for streptococcal pharyngitis: evaluation of sensitivity with respecttotrueinfections. J. Pediatr. 108:654-658.

6. Gerber, M. A., M. F. Randolph, and D. R. Mayo. 1988. The group A streptococcal carrier state. Areexamination. Am. J. Dis. Child. 142:562-565.

7. Kaplan, E. L., R. Couser,B. B.Huwe, C. McKay, andL. W.

Wannamaker. 1979. Significance ofquantitative salivary

cul-turesfor groupAand non-group AP-hemolyticstreptococci in

patients with pharyngitis and in their familycontacts.Pediatrics 64:904-912.

8. Kaplan, E. L., F. H. Top, Jr., B. A. Dudding, and L. W. Wannamaker. 1971. Diagnosis of streptococcal pharyngitis: differentiation of active infection from the carrier state in the symptomatic child. J. Infect. Dis. 123:490-501.

9. Kellogg,J.A. 1990.Suitability of throat culture procedures for detection of group A streptococci andasreference standards for evaluation of streptococcal antigen detection kits. J. Clin. Microbiol. 28:165-169.

10. Kellogg, J. A., R. C. Landis, A. S. Nussbaum, and D.A.

Bankert.1987. Performance ofanenzymeimmunoassay system

and anaerobic culture for detection ofgroup Astreptococci ina pediatrics practice versus a hospital laboratory. J. Pediatr. 111:18-21.

11. Kellogg, J. A., and J. P. Manzella. 1986. Detection ofgroup A streptococci in the laboratory orphysician's office. Culturevs antibody methods. JAMA 255:2638-2642.

12. Meyer, R. J.,and R.J. Haggerty.1962.Streptococcal infections infamilies. Factors altering individualsusceptibility. Pediatrics 29:539-549.

13. Moffet, H. L. 1975. Pediatric infectious diseases: a problem-oriented approach, p. 15-49. J. B. Lippincott, New York. 14. Packer, H.,M. B.Arnoult,andD. H. Sprunt. 1956. Astudy of

hemolytic streptococcal infections in relation to antistreptolysin Otiterchanges in orphanage children.J. Pediatr. 48:545-562. 15. Roddey, 0. F., H. W. Clegg, L. T. Clardy, E. S. Martin, and

R. L. Swetenburg. 1986. Comparison of a latex agglutination

test and four culture methods for identification of group A streptococci inapediatric office laboratory. J. Pediatr. 108:347-351.

16. Wannamaker, L. W. 1979. Changes and changing concepts in thebiology of groupAstreptococci and in theepidemiology of streptococcal infections. Rev. Infect. Dis. 6:967-973.

James A. Kellogg

Clinical Microbiology Laboratory York Hospital