Vol. 11, No. 3JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1980, p. 249-2550095-1 137/80/03-0249/07$02.00/0

Evaluation of Three Commercially Available Test Productsfor Serogrouping Beta-Hemolytic Streptococci

M. SLIFKIN* AND GAIL R. POUCHET-MELVINDepartment of Laboratory Medicine, Section ofMicrobiology, Allegheny General Hospital, Pittsburgh,

Pennsylvania 15212

Three beta-streptococci serogrouping kits, Phadebact, SeroSTAT, and Strep-tex, were evaluated as to their sensitivity, accuracy, and suitability as methodsfor serogrouping streptococci in a clinical microbiology laboratory. The majorityof the primary isolates examined by the various methods associated with each ofthe three kits were correctly identified. The Streptex direct mixed-culture pro-

cedure was more often associated with the observation of cross-reactivity thanwith the direct procedures of the other two kits which did not employ mixedgrowth cultures. Furthermore, the Streptex kit was associated with more false-negative responses than those determined by the other two kits under evaluation.These results appeared to be due to the relatively poor sensitivity of the Streptexgrouping reagents. The Streptex test procedures required more labor than theother kit procedures, requiring a 1-h enzymatic extraction step for the release ofthe group antigens. The SeroSTAT kit provided only a direct procedure and,thus, is limited in its application. The Phadebact procedures were the mostversatile by providing not only a direct and a 24-h grouping procedure, but alsoby including a 4-h method that may be employed as required by the clinicalmicrobiologist.

The coagglutination method was previouslyshown to be an accurate, rapid, and simplemethod (1, 2, 6, 10, 12, 13, 16, 17) for the sero-grouping of beta-hemolytic streptococci. Thismethod employs, in part, Cowan I staphylococcithat are coated with rabbit gamma globulin spe-cific for a streptococcal group. The adsorbedantibody has its Fc portion attached to thestaphylococcal protein A and its antigen-com-bining Fab parts directed outward (4).

Kits for serogrouping various beta-hemolyticstreptococci by coagglutination are commer-cially available (Phadebact Streptococcus Testkit, Pharmacia Diagnostics). Recently, it wasshown that latex particles sensitized with rabbitgamma globulin specific for various streptococcicould be employed in the serogrouping of thesebacteria (14). At present, two commerciallyavailable latex agglutination kits for serogroup-ing these bacteria are available (SeroSTAT,Scott Laboratories, Inc.; Streptex, Weilcome Re-search Laboratories) for use in the clinical mi-crobiology laboratory. Materials for various pro-cedures including direct, nonmixed, directmixed, and 4-h and 2-h cultures, as well as formixed cultures, are included in the respectivecoagglutination and latex agglutination kits. Ac-cordingly, the purpose of this investigation wasto evaluate the serogrouping reagents from thesethree commercially available kits as to their

sensitivity, accuracy, and suitability as methodsfor serogrouping streptococci in a clinical labo-ratory.

MATERIALS AND METHODSOrganisms. The cultures of bacteria were obtained

from clinical specimens and stock cultures. Stock cul-tures of serogrouped beta-hemolytic streptococci weremaintained in Trypticase (BBL Microbiology Sys-tems) soy broth containing 15% (vol/vol) glycerol at-70°C (18). Clinical specimens received on transportswabs (Culturette, Marion Scientific Corp.) and thestock cultures were streaked on Columbia sheep bloodagar plates (BBL) to obtain isolated organisms. Theplates were incubated at 35°C under anaerobic con-ditions in GasPak (BBL) units for 12 to 18 h. Theplates were then observed for the presence of beta-hemolytic streptococci with the application of Gramstaining and the determination of hemolytic and cat-alase activity (7, 8, 20).

Isolated colonies of beta-hemolytic streptococcifrom each clinical primary plate were selected andemployed for the direct-plate coagglutination methodwith the Phadebact, Streptex, and SeroSTAT re-agents. However, approximately 40% of the beta-he-molytic streptococci employed for the direct-plate testwith the Streptex reagents were obtained as mixedcultures by streaking a loop across the path of thestreptococci. One isolated colony from each clinicalprimary plate was selected and inoculated into 2 ml ofTodd-Hewitt broth (BBL) for use in the respective 4-(9, 16) and 24-h Phadebact and 24-h Streptex methods.

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Similarly, isolates were inoculated into 40 ml of Todd-Hewitt broth manufactured by BBL and Difco Labo-ratories and were examined for cross-reactivity by thevarious serogrouping reagents.

Primary isolates (327) of beta-hemolytic strepto-cocci were derived from the following locations (num-ber of isolates in parentheses): upper (143) and lower(77) respiratory tract, blood (26), wounds (70), urine(59), and the umbilicus (2). These 12- to 18-h primaryisolates were serogrouped by all three kits with theirassociated procedures when possible. The results werecompared with those obtained by the autoclave ex-

traction method.Grouping of streptococci by coagglutination.

The Phadebact Streptococcus Test kit containing re-

agents for serogrouping groups A, B, C, and G strep-tococci was employed following the manufacturer'sprotocol for the direct-plate, 4-, and 24-h procedures.At least five isolated colonies from each clinical pri-mary plate were used when applying the direct-plateprocedure (17).Grouping of streptococci by latex agglutina-

tion. The SeroSTAT Streptococcus Test was em-

ployed following the method as described in the kit.This method employed only isolated colonies from an

isolation medium and thus is classified in this investi-gation as a direct-plate method. The Streptex proto-cols presented in the kit permit a 24-h test and a

direct-plate method that permits the use of either a

pure culture of isolated colonies of beta-hemolyticstreptococci or a mixed culture obtained by one or

more passes of a loop across the surface of a primaryplate containing beta-hemolytic streptococci. After 1h, an enzyme extraction step employing pronase was

used with the direct and 24-h Streptex methods.Lancefield grouping. Extracts were prepared for

the Lancefield grouping by the autoclave extractionmethod (15). Commercial antisera (Burroughs Well-come Co.) for groups A, B, C, D, F, and G were

employed in the performance of the precipitin test incapillary tubes (19). All the grouping methods were

performed blind, relative to each other.

Several approaches were utilized to determine thesensitivity of each kit in the ability to sero-react withbeta-hemolytic streptococci. Strains representinggroup A streptococci were washed twice in sterile HAbuffer (Difco) and suspended in 1.0 ml of HA buffer.The suspensions were diluted in twofold steps andtested for coagglutination or agglutination with theserogroup A reagent from each of the three kit sys-

tems. The strength of reactivity was observed as wellas the time in seconds required for the response tooccur. Plate counts of each dilution of bacteria were

prepared with a calibrated 0.01-ml loop. In other ex-

periments, various numbers of group A colonies were

picked with an inoculating needle to determine theminimal number of colonies required for a serogroup-

ing response.

RESULTS

Accuracy of reagents. The comparative re-

sults of the three serogrouping procedures areshown in Table 1. The direct-plate Phadebactprocedure, when compared to the precipitinmethod, permitted the correct identification of86 primary beta-hemolytic isolates from the 87isolation plates (98.9%). A minimum of five col-onies was required for testing with each of thefour Phadebact reagents. The 4- and 24-h Phad-ebact readings yielded results that correspondedin all cases (100% correct) to those obtained bythe Lancefield method.The direct-plate SeroSTAT method yielded

results that correctly identified 166 of 167(99.4%) isolates, compared to the referencemethod. However, due to the lack of a 24-hmethod, not all of the isolates tested by theother two kits could be identified.The direct-plate Streptex method was associ-

ated with the misidentification of 15 beta-he-molytic streptococcal isolates (95.4% correct).

TABLE 1. Comparison of three test products and associated methods for the serogrouping of beta-hemolyticstreptococci from primary isolation plates

Phadebact procedures SeroSTAT pro- Streptex procedurescedure Lance-field

Direct 4-h 24-h (direct) Direct' 24-h pro-Group cedureb

No. No. No. No. No. No. (no.No. cor- No. cor- No. cor- No. cor- No. cor- No. cor- group-

tested rectly tested rectly tested rectly tested rectly tested rectly tested rectly ed)grouped grouped grouped grouped grouped grouped

A 35 34 97 97 127 127 67 67 101 95 127 127 127B 48 48 149 149 153 153 80 80 133 133 153 153 153C 2 2 19 19 22 22 11 10 19 12 22 21 22D NTe NT NT NT NT NT NT NT 40 40 40 40 40F NT NT NT NT NT NT NT NT 10 8 10 9 10G 1 1 12 12 12 12 7 7 19 19 19 19 19Non- 1 1 5 5 6 6 3 3 4 4 6 6 6grouped

Isolated colonies or mixed cultures containing beta-hemolytic colonies were employed.Rantz-Randal autoclave extraction method.NT, Not tested; kit did not provide reagent to serogroup D or group F streptococci.

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These misidentifications were due, in part, tothe lack of responsiveness of five group A iso-lates, and false-positive reactivity with the groupC serogrouping reagent. The latter cross-reactiv-ity was due to the presence of Streptococcuspneumoniae and other bacteria collected on theloop with the mixed-culture procedure, as willbe discussed in the section on cross-reactivity.Furthermore, two group A isolates gave equiv-ocal agglutination responses with the group Creagent. The seven misidentified group C iso-lates and one group F isolate did not react withtheir respective serogrouping reagents. Anothergroup F isolate yielded a cross-reactive responsewith the group C reagent with the mixed-culturemethod, whereas another isolate did not agglu-tinate with the specific reagent. Two nongroup-able strains also yielded cross-reactive aggluti-nation with the group C reagent with the directmixed-culture method. Two misidentificationsoccurred with the 24-h Streptex method (99.5%correct). These were due to the cross-reactivityof a group C isolate with group A reagent andthe false-negative response of a group F isolatewith the specific serogrouping reagent.

Sensitivity of reagents. Approximately40,000 to 60,000 colony-forming units combinedwith both Phadebact and SeroSTAT reagentsto cause a minimal response, whereas 80,000 to100,000 colony-forming units resulted in a strongserogrouping response. On the other hand, theminimal number of colony-forming units tocause a latex agglutination response with theStreptex group A reagent was 80,000 colony-forming units (Table 2). Both the Phadebactand SeroSTAT group A reagents reacted withfive colonies to form a weak response with 1 minof observation. The application of 10 or morecolonies with either of these two reagents, how-ever, resulted in stronger reactions within 30 sor less. The enzyme-extraction procedure of theStreptex method required 10 colonies for a min-imal latex agglutination reaction, whereas 20 ormore colonies yielded relatively stronger agglu-tination responses (Table 3).A third experiment was employed to further

pursue the sensitivity of the three kit reagentsfor the serogrouping of group A and group Cstreptococci. The polyvalent control antigenfrom a Streptex kit was diluted in twofold steps,and one drop of each dilution was mixed, re-spectively, with one drop of group A and groupC reagent from each of the three streptococcalserogrouping test kits. Both the Phadebact andSeroSTAT reagents responded similarly by pro-ducing their coagglutination or agglutination re-

sponses with their group A reagents through a1:64 dilution ofpolyvalent antigen. Furthermore,

EVALUATION OF SEROGROUPING KITS 251

TABLE 2. Minimal number of colony-forming unitsrequired for coagglutination and agglutination ofgroup A streptococci with Phadebact, SeroSTAT,

and Streptex reagents

Number of col- Response with group A reagentaony-forming

units Phadebact STrAT Streptex

1,000 - - -10,000 - - -40,000 + + -60,000 + + -80,000 ++ ++ +100,000 ++ ++ ++

a +, Weak response; -, no response; ++, moderateresponse; +++, strong response.

TABLE 3. Minimal number ofgroup A streptococcicolonies required for coagglutination and

agglutination with three commercially availableserogrouping kitsa

No. colonies Phadebact SeroSTAT Streptex

3 _b5 + + _8 + + _10 ++ +++15 +++ +++-20 +++ +++ +25 +++ +++ ++30 +++ +++ +++

a Twelve-hour colonies grown on sheep blood agarat 350C.

b-, No response; +, weak response; ++, moderateresponse; +++, strong response.

the periods of time for the reactions to occurthrough the various dilutions of polyvalent an-tigen were quite similar. The Phadebact groupC reagent also produced observable coagglutin-ation responses through a 1:64 dilution, whereasthe SeroSTAT group C reagent yielded slightlyslower responses, especially at the higher dilu-tions of the polyvalent antigen. In contrast tothe latter two reagents, the response time ofagglutination with the group A and group CStreptex reagents was considerably slower. Also,the greatest dilution at which detectable agglu-tination was evident occurred at a 1:8 dilution ofthe polyvalent antigen.Twenty colonies each of four group A strep-

tococci stock strains derived from the AmericanType Culture Collection and three clinical iso-lates of group A streptococci were serogroupedby each of the three products to determine theresponse time with the group A reagents foreach of the three kits. The time for serogroupingresponse by both the Phadebact and SeroSTATreagents for these seven strains of group A strep-tococci was similar and occurred between 6 and

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252 SLIFKIN AND POUCHET-MELVIN

10 s. The response time with the Streptex re-agents, employing the enzyme extract from eachof these bacterial strains, was consistently pro-longed. The latter agglutination reactions oc-curred between 16 and 21 s.

Cross-reactivity. The potential for cross-reactivity of the serogrouping reagents fromeach of the three kits was determined for 13species of bacteria including five members of theEnterobacteriaceae, Pseudomonas aeruginosa,Staphylococcus aureus, and five species of viri-dans streptococci. The Phadebact group C re-agent cross-reacted with all of the strains ofKlebsiella pneumoniae and S. pneumoniae ex-amined. The reaction time for the coagglutina-tion response with S. pneumoniae was quiterapid in contrast to that observed for K. pneu-moniae. The cross-reactivity responses for theStreptex reagents were similar to those of thePhadebact reagents except that the group Areagent also produced agglutination with all thetest strains of K. pneumoniae. Cross agglutina-tion with the SeroSTAT reagents occurred withK. pneumoniae and S. aureus with the group A,B, C, and G reagents at just under 40 s ofobservation. The group C reagents from theSeroSTAT Kit reacted with all the S. pneumo-niae strains (Table 4). The group A reagents ofthe Phadebact and SeroSTAT kits were shownto cross-react with drops of Todd-Hewitt brothmanufactured by Difco but not with the BBLproduct (Table 5).

Since the manufacturer of Streptex stated inthe protocol of the kit that mixed growth maybe employed for the serogrouping of beta-strep-tococci from a culture plate, the possibility of

TABLE 5. Cross-reactivity response of Phadebact,SeroSTAT, and Streptex group A reagents with

BBL and Difco Todd-Hewitt broth

Todd-Hewitt broth Phade- Sero- Strep-Todd-ewittbroth bact STAT tex

BBL (lot no. EIDHRS) -a

BBL (lot no. EIDHRT)Difco (lot no. 653123) + +Difco (lot no. 654930) + +

a -, No response; +, weak response.

cross-reactivity problems with other bacteriawith this procedure was examined. Sweeps weretaken from 10 primary plates containing 5 to 200beta-hemolytic colonies. The agglutination withthe direct mixed-culture method from eight ofthe plates containing group A streptococci wassomewhat ambiguous since a response was ob-served with both the group A and group CStreptex reagents. Furthermore, the time of theagglutination response was only slightly fasterwith the group A reagent than that with thegroup C reagent for four of these plates andessentially the same with the other four plates.Another plate containing 80 beta-hemolytic col-onies of group A streptococci did not respondwith the group A reagent when approximately20 colonies were collected with a sweep of theloop and extracted for the serogrouping proce-dure. A sweep of colonies from two primaryplates containing only two and five colonies ofgroup A streptococci, respectively, yielded onlyfalse-positive group C responses. On subculture,these organisms were correctly serogrouped bythe Streptex group A reagent (Table 6).

DISCUSSIONTABLE 4. Cross-reactivity of commercial

streptococcus agglutination grouping reagents withvarious bacteria

Cross-reactive reagents

Organism Phade- SeroSTAT Streptex

bact Ca A B C G A C

K. pneumoniae +C + + + + + +(10)b

S. aureus (10) - + + + + - -

S. pneumoniae + - - + - - +(10)

All others (53)da Phadebact reagents A, B, and G and Streptex B,

D, F, and G did not react with any of the bacteriatested.

b Number of strains tested.c +, Weak response; -, no response.d Other bacteria tested: Citrobacter diversus (5),

Enterobacter cloacae (10), Escherichia coli (10), P.aeruginosa (10), Serratia liquefaciens (10), viridansstreptococci (8).

Numerous investigations have shown thatcoagglutination testing, particularly with thePhadebact Streptococcus Test, affords the clin-ical microbiologist a rapid and reliable methodto serogroup beta-hemolytic streptococci (10, 16,17). The present investigation confirms the di-agnostic potential of this method and shows thecapabilities of the relatively new SeroSTAT andStreptex latex agglutination tests for the sero-grouping of beta-hemolytic streptococci.This work substantiates our previous report

(7) which showed that the Phadebact test sys-tem permits the serogrouping of colonies takendirectly from the surface of a primary isolationplate when at least five beta-hemolytic strepto-cocci are present. These results are in contrastto a recent report stating essentially that nodirect-plate method exists for the PhadebactStreptococcus Test (13). Likewise, as previouslyreported (17), when sufficient numbers of thesecolonies are not available for this direct proce-

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TABLE 6. Cross-reactivity of response with Streptexreagents using mixed-culture plate procedure with

bacteria from primary throat culture platesNo. of

beta-he-Plate byta Streptex re- Final identifica-no. molytic sponsea tionbcolonies

on plate

1 5 C(10) Beta-hemoly-sis due to S.pneumoniae

2 30 A(12), C(10) Group A3 2 C(10) Group A4 80 c Group A5 135 A(15), C(25) GroupA6 147 A(11), C(15) Group A7 159 A(10), C(20) GroupA8 170 A(18), C(18) Group A9 175 A(20), C(19) Group A10 200 A(8), C(15) Group Aa Time of agglutination response (seconds) in paren-

theses.Rantz-Randal extraction.e, No reactivity with any serogrouping reagent.

dure or when the possibility exists of pickingcolonies of other species of bacteria with theloop, either the 4- or 24-h Phadebact proceduremay be utilized. Furthermore, an earlier inves-tigation has shown the coagglutination proce-dure to be applicable to serogrouping strepto-cocci directly on the colonies growing on primaryplates (6).There is only one procedure for the Sero-

STAT kit and this one, like that of the Phade-bact direct method, employs colonies from a

primary plate or from a pure culture obtainedby subculture. Furthermore, its sensitivity andspecificity response with a minimum of five col-onies was similar to that observed with the di-rect-plate Phadebact procedure. In contrast tothese two kits, the Streptex procedure requireda greater number of bacterial colonies with itsrequired extraction step to visualize its aggluti-nation response. Not all primary isolation platesmay contain the minimal number of coloniesrequired for the seroidentification of strepto-cocci with each of the respective kits. This de-pendence upon minimal numbers of isolatedstreptococcal colonies may present some limi-tations in the application of these kits. In theseinstances, the 4-h broth-culture procedure maybe employed with the Phadebact reagents, or asubculture with a blood agar plate may be usedto seroidentify the isolate.The major discrepancies that were detected

with the Streptex kit appear to be related to theuse of the mixed-culture method. This methodappears to be associated with cross-reactivitybetween the Streptex group A and group C

EVALUATION OF SEROGROUPING KITS 253

reagents and other bacteria collected in thesweep across an isolation plate. Furthermore,the relatively slower reactivity of its serogroup-ing reagents in conjunction with their cross-re-active potential resulted in the misidentifica-tions or equivocal serogrouping responses ob-served in this investigation. Agglutination re-sponses occurring from 1 to 2 min with Streptexwere recently reported (K. C. Gross, M. P.Houghton, and L. B. Senterfit, Abstr. Annu.Meet. Am. Soc. Microbiol. 1979, C170, p. 338).The coagglutination of all the strains of S.

pneumoniae tested in this investigation with thegroup C reagents from each of the three sero-grouping kits is consistent with the fmdings ofother investigators (3, 10, 11, 14). The cross-reactivity of this bacteria with the Phadebactand SeroSTAT serogrouping reagents would notcause any difficulties due to the use of onlyisolated colonies with these two methods. Thisis in contrast to the Streptex kit (i.e., 4.6% misi-dentification) which permits the application ofmixed as well as pure cultures of beta-strepto-cocci by direct serogrouping. When employingany of these kits in the clinical laboratory, it isimperative to screen out pneumococci throughcareful examination of the morphology and he-molytic activity of the bacterial colonies. Thisprotocol will reduce the cross-reactive potentialfor error with all these kits regarding the pneu-mococci (Table 4). This potential was minimalwith the Phadebact and SeroSTAT serogroup-ing reagents because the manufacturer suggeststhe use of isolated colonies. In contrast, theStreptex kit permits the application of bothmixed and pure culture of beta-streptococci bydirect serogrouping.

Certainly, the lower sensitivity of the Streptexserogrouping reagents was evident in this inves-tigation. When compared to the Phadebact andSeroSTAT procedures, the Streptex proceduresrequired extra labor, particularly a 1-h extrac-tion step before using the latex agglutinationreagents. Furthermore, the Streptex extractionmethod, as borne out in this investigation, maynot yield enough group antigen for a serogroup-ing response and, thus, was frequently associatedwith false-negative responses more than witheither the Phadebact or SeroSTAT kit. This wasthe case when fewer than 20 colonies were usedfor an extraction. A possible explanation forthese negative responses with the Streptex kitmay be associated with the vagueness of theexplanation of its package insert concerning thedensity of the bacterial suspension required forthe pronase extraction. Extraction enzymes suchas Pronase B have been shown to be effectivefor the preparation of group-specific streptococ-cal antigens (5). However, this procedure re-

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254 SLIFKIN AND POUCHET-MELVIN

quires the use of chemically clean glassware anddouble-distilled water for an unimpeded extrac-tion (21).Thus, although the Streptex test system, un-

like the Phadebact and SeroSTAT systems, per-

mits serogrouping group D and group F beta-streptococci, it is recommended that alternativetests be additionally employed if the beta-he-molytic streptococci are other than group A, B,C, or G. Namely, group D beta-streptococci maybe identified with the use of bile-esculin agar (8).At present, except for their relatively small col-ony size, no biochemical nor physiological testexists for the identification of group F beta-streptococci (7, 8). Therefore, the precipitationmethod employing antisera to group F carbo-hydrate should be utilized if desired by the lab-oratory.Due to experience in the recognition of the

morphology and type of beta-hemolysis of thestreptococcal colonies as well as their source,particularly with the group A and group B iso-lates, we often initially employed the groupingreagents for these beta-hemolytic streptococciwith certain isolates. Furthermore, these twoserogroups were isolated in greater numbersthan the other serogroups. Accordingly, it wasconsidered a disadvantage, in contrast to thePhadebact and SeroSTAT kits, not to have sep-arate commercial serogrouping Streptex re-

agents available. One SeroSTAT dispenser oflatex grouping reagent became blocked with la-tex particles so that release of reagent was

impeded. We solved this problem by openingthe orifice with a pin. Loss of reactivity of one

vial of group A Streptex reagent for group Astreptococci was observed after 6 months ofstorage at 10°C. Neither of these difficulties wasnoted for the Phadebact kit, although slightleakage from the dispensing vials occasionallyoccurred. We solved this problem by lightlytapping the vial on the countertop to remove

reagent from the cap.As with the SeroSTAT kit, the Phadebact kit

does not require an enzymatic procedure forextraction of the group antigens. The Phadebactprocedures are more versatile than those asso-

ciated with the other two kits by providing notonly a direct-plate and 24-h protocol, but also a

4-h method that may be employed as requiredby the clinical microbiologist.The cross-reacting nature of Todd-Hewitt

broth manufactured by Difco was observed inthis investigation. Thus, it became evidence thatthe 4- and 24-h Phadebact procedures should beemployed only with the BBL product as rec-

ommended by the manufacturers.In summary, all three kits evaluated in this

J. CLIN. MICROBIOL.

investigation permitted the correct serogroupingof the majority of the clinical isolates of beta-streptococci examined. In contrast to the direct-plate procedure, the Streptex kit offers both adirect-plate procedure on isolated as well as onmixed growth cultures. However, the Streptexdirect-plate procedure on mixed cultures wasmore often associated with cross-reactivity. Fur-thermore, the Streptex test was associated withmore false-negative responses than that ob-served with the Phadebact and SeroSTAT re-agents. It was concluded that the PhadebactStreptococcus Test was the most proficientthrough the availability of its three proceduresof serogrouping that include direct, 4-h, and 24-h, procedures.

LITERATURE CITED1. Arvilommi, H. 1976. Grouping of beta-hemolytic strep-

tococci by using coagglutination, precipitation or baci-tracin sensitivity. Acta Pathol. Microbiol. Scand. Sect.B 84:79-84.

2. Arvilommi, H., 0. Uurasmaa, and A. Nurkkala. 1978.Rapid identification of group A, B, C, and G beta-haemolytic streptococci by a modification of the co-agglutination technique. Comparison ofresults obtainedby co-agglutination, fluorescent antibody test, counter-immunoelectrophoresis, and precipitin technique. ActaPathol. Microbiol. Scand. Sect. B 86:107-111.

3. Austrian, R., C. Buettger, and M. Dale. 1972. Problemsin the classification and pathogenic role of alpha andnonhemolytic streptococci of the human respiratorytract, p. 355-370. In L. W. Wannamaker and J. M.Matsen (ed.), Streptococci and streptococcal disease.Academic Press Inc., New York.

4. Christensen, P., G. Kahlmeter, S. Jonsson, and G.Kronvall. 1973. New method for the serological group-ing of streptococci with specific antibodies adsorbed toprotein A-containing staphylococci. Infect. Immun. 7:881-885.

5. Ederer, G. M., M. M. Herrmann, R. Bruce, J. M.Matsen, and S. S. Chapman. 1972. Rapid extractionmethod with Pronase B for grouping beta-hemolyticstreptococci. Appl. Microbiol. 23:285-288.

6. Edwards, E. A., and G. L Larson. 1974. New methodof grouping beta-hemolytic streptococci directly onsheep blood agar plates by coagglutination of specifi-cally sensitized protein A-containing staphylococci.Appl. Microbiol. 28:972-976.

7. Facklam, R. R. 1974. Streptococci, p. 96-108. In E. H.Lennette, E. H. Spaulding, and J. P. Truant (ed.),Manual of clinical microbiology, 2nd ed. American So-ciety for Microbiology, Washington, D.C.

8. Facklam, R. R., J. F. Padula, L. G. Thacker, E. C.Wortham, and B. J. Sconyers. 1974. Presumptiveidentification of group A, B, and D streptococci. Appl.Microbiol. 27:107-113.

9. Farrell, B., and I. Amirak. 1976. Agglutination groupingof streptococci. (Letter to the editor.) Lancet ii:1082.

10. Finch, R. G., and I. Phillips. 1977. Serological groupingof streptococci by a slide coagglutination method. J.Clin. Pathol. 30:168-170.

11. Hahn, G., and L. Nyberg. 1976. Identification of strep-tococcal groups A, B, C, and G by slide co-agglutinationof antibody-sensitized protein A-containing staphylo-cocci. J. Clin. Microbiol. 4:99-101.

12. Kirkegaard, M. K., and C. R. Field. 1977. Rapid slidecoagglutination test for identifying and typing group B

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streptococci. J. Clin. Microbiol. 6:266-270.13. Lrm, D. V., R. D. Smith, and S. Day. 1979. Evaluation

of an improved rapid coagglutination method for theserological grouping of beta-hemolytic streptococci.Can. J. Microbiol. 25:40-43.

14. Lue, Y. A., I. P. Howitt, and P. D. Ellner. 1978. Rapidgrouping of beta-hemolytic streptococci by latex agglu-tination. J. Clin. Microbiol. 8:326-328.

15. Rantz, L. A., and E. Randall. 1955. Use of autoclavedextracts of haemolytic streptococci for serologicalgrouping. Stanford Med. Bull. 13:290-291.

16. Rosner, R. 1977. Laboratory evaluation of a rapid four-hour serological grouping of groups A, B, C, and G beta-streptococci by the Phadebact Streptococcus Test. J.Clin. Microbiol. 6:23-26.

17. Slifkin, M., C. Engwall, and G. R. Pouchet. 1978.Direct-plate serological grouping of beta-hemolyticstreptococci from primary isolation plates with the

EVALUATION OF SEROGROUPING KITS 255

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