Counterimmunoelectrophoresis - Journal of Clinical Microbiology

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Vol. 1, No. 2 Printed in U.S.A.

JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1975, p. 188-191 Copyright 0 1975 American Society for Microbiology

Rapid Identification of Group B Streptococci by Counterimmunoelectrophoresis HARRY R. HILL,* MARY E. RITER, SUE K. MENGE, DWIGHT R. JOHNSON, AND JOHN M. MATSEN Departments of Pathology* and Pediatrics, University of Utah College of Medicine, Salt Lake City, Utah 84132, and the Departments of Clinical Laboratory Medicine and Pediatrics, University of Minnesota, Minneapolis, Minnesota 55455 Received for publication 7 October 1974

Beta-hemolytic streptococcal isolates have been examined by counterimmunoelectrophoresis (CIE) with group B antiserum to determine whether this technique is of value in the rapid identification of group B strains. Ninety stock cultures and 100 clinical isolates of beta-hemolytic streptococci including representatives of groups A, D, C, G, and B were inoculated into Todd-Hewitt broth; after incubation at 37 C for 1, 2, 3, and 4 h, aliquots of the whole broth cultures were removed and tested by CIE. Antigen was not regularly detected in the 1-, 2-, and 3-h samples, but after 4 h all 126 group B streptococcal strains identified by the capillary precipitin reaction gave CIE precipitin bands with group B antiserum. None of the 58 non-group B strains gave precipitin reactions with this antiserum. Cerebrospinal fluid from an infant with group B streptococcal meningitis and peritoneal fluid from a patient with group B streptococcal peritonitis had free group B antigen detected by the CIE technique. CIE of broth cultures and direct body fluids appears to be a rapid and sensitive method for the identification of group B streptococcal strains. Streptococci of Lancefield group B represent a significant hazard to parturient women and neonates where colonization may be associated with abortion, stillbirth, prematurity, and fulminating meningitis or sepsis (1, 6, 9). Epidemiological studies (1, 8) have indicated that 4 to 25% of pregnant females are colonized with group B streptococci at delivery and that neonatal sepsis due to this agent occurs in as many as 2 per 1,000 live births (8). An early septicemia form of the disease with infection acquired at or before delivery has been described, which is often fatal within 24 to 48 h (2, 8). In addition, a late onset meningitis form possibly resulting from nosocomial infection has been observed (2, 8). A rapid means of identifying group B streptococci would be of significant benefit to the clinician and to the hospital epidemiologist. The present report describes the results of counterimmunoelectrophoresis (CIE) studies for rapid detection and identification of clinical isolates of group B streptococci. (This work was presented in part at the Annual Meeting of the American Society for Microbiology, Chicago, Ill., 12 to 17 May 1974.) 188


Dacron swabs obtained from clinical sources (including the throat, skin, and urogenital tract of neonates and pregnant females; wounds; and throat cultures from patients with pharyngitis) were used to inoculate 5c. sheep blood agar plates. After incubation for 12 to 18 h at 37 C, beta-hemolytic streptococcal colonies were identified and one to three isolated colonies were subcultured in 1-ml aliquots of ToddHewitt broth (Difco). Stock strains of beta-hemolytic streptococci in blood broth were similarly inoculated into 1-ml aliquots of Todd-Hewitt broth. Body fluids were obtained by percutaneous needle aspiration and tested immediately. No extraction procedure (i.e., acid, Pronase) was used in the CIE studies. Rabbit antiserum against the group B streptococcal polysaccharide-grouping antigen was obtained from the Center for Disease Control, Atlanta, Ga., and Burroughs-Wellcome Laboratories, Beckenham, England. Rabbit antisera against the type-specific antigens of group B streptococci including types Ia, Ib, Ic, II, and HI were kindly supplied by Rebecca Lancefield of the Rockefeller Institute. CIE was carried out on glass microscope slides (1 by 3 inch [2.54 by 7.62 cm]) covered with 3 ml of 1% agarose (Fisher Scientific Co.) in barbital buffer (pH 8.8; Gelman). Parallel rows of 2-mm diameter wells were cut in the agar so that 2 mm separated the

VOL. 1, 1975

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antigen and antiserum wells. The antiserum, in 10-Al aliquots, was added to the well nearest the anode, whereas the antigen (body fluids, broth culture) was added in similar volumes to the well nearest the cathode. Electrophoresis was carried out at room temperature for 30 min using 5 to 7 mA per slide in a Gelman electrophoresis apparatus. Strips of Whatman no. 1 filter paper were used for electrode wicks; barbital buffer (pH 8.8; Gelman) was placed in the electrophoresis chamber. All beta-hemolytic streptococcal strains were serologically grouped by the Lancefield capillary precipitin technique (11). In addition, several group B strains were typed by the capillary precipitin method (12, 13).

RESULTS A heavy line of precipitation developed when 4-h broth cultures of group B streptococci were electrophoresed against group B antiserum (Fig. 1). No precipitation was observed with strains of other groups. Aliquots of broth cultures of 16 group B strains were examined after 1, 2, 3, and 4 h of incubation at 37 C. None of the strains gave a positive reaction after 1 h of incubation, but by 4 h all could be identified as group B strains (Table 1). Additional experiments indicated that heavy suspensions of group B organisms made from colonies on blood agar plates did not give precipitin reactions without incubation. Moreover, membrane filtration of positive broth cultures did not remove the precipitable material, suggesting that free group B antigen in the fluid was necessary for a positive reaction to develop. Ninety stock strains of beta-hemolytic streptococci that had been identified by the capillary precipitin technique of Lancefield (11) were inoculated in 1-ml aliquots of Todd-Hewitt broth and tested by CIE against group B antiserum after 4 h of incubation. Thirty-seven strains were identified as group B streptococci by the capillary precipitin reaction; all 37 reacted with group B antiserum in the CIE method (Table 2). None of the 27 group A, 10 group C, 13 group G, or 3 group D strains gave reactions with the B antiserum. One hundred clinical isolates of betahemolytic streptococci that were fluorescentantibody negative for group A organisms were tested by CIE with group B antiserum. All of the eighty-nine strains that were identified as group B organisms by the Lancefield technique were also positive by CIE with group B antiserum. The 11 isolates of non-group B streptococci gave no reaction with this antiserum (Table 3).





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FIG. 1. Precipitin lines produced by CIE of broth cultures of group B streptococci and group B antiserum. AS, Antiserum well; AG, antigen (culture filtrate) well; B, group B streptococcal; G, group G


Cerebrospinal fluid and peritoneal fluid from an infant with meningitis and an infant with peritonitis due to group B streptococci were placed directly into the antigen well. Both gave precipitin lines with the group B antiserum after electrophoresis for 30 min. The serum from a patient with group B streptococcal sepsis did not, however, give a posivite reaction when




TABLE 1. CIE of sequential broth cultures of group B streptococci Incubation time (h) No. positive by CIE


2 3 4

0 1 4 11

Cumulative 0

6 31 100

TABLE 2. CIE of 90 stock strains of streptococci with group B antisera Lancefield capillary precipitin reaction

No. strains tested

No. positive by CIE with group B antisera

Group B Group A Group C Group G GroupD

37 27 10 13 3

37 0 0 0 0

added to the antigen well. It was possible to identify free group B streptococal antigen by CIE in broth taken from overnight blood cultures of two patients with group B streptococcal sepsis.

Twenty strains of group B streptococci including members of types Ia, Ic, II, and II were divided into serological types by using the Lancefield capillary precipitin reaction (12, 13) and CIE. (Typing antiserum of sufficient reactivity was not available for type lb.) Type II and type III strains could be positively identified after 4 h of incubation in broth. There were cross-reactions observed between type Ia and type Ic strains as expected, but each reacted with the homologous type I antiserum (Table 4). DISCUSSION CIE has been used in the rapid diagnosis of meningitis and sepsis due to Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae (5, 7, 10). More recently, Dajani (4) used this technique to identify streptococcal grouping antigen in acid extracts of growth taken directly from blood agar plates. Group A antigen was detected in extracts from 69 of 85 group A strains, group C antigen was detected in 9 of 9 group C strains, and group G antigen detected in 14 of 14 group G strains. Antigen could be detected in acid extracts of only 17 of the 31 group B strains tested, howwas


In the present report all group B streptococcal strains could be postively identified after incu-

bation in broth for 4 h. Strains of other groups of beta-hemolytic streptococci did not give reactions with the group B antiserum. The antigen that reacts in the CIE procedure appears to be unassociated with whole organisms in the broth culture since membrane filtration did not prevent the precipitin line from developing. Moreover, it appears that a certain period of incubation is required before enough free antigen is present to give the reaction. Although only three body fluids were examined for group B antigen directly by the CIE procedure, two, cerebrospinal fluid and peritoneal fluid, were found to have free antigen. This suggests the potential for positively identifying infections due to group B streptococcal organisms within a 30-min period. We have also been successful in detecting free antigen in 4-h broth cultures of swab taken from patients with group B streptococcal colonization. Additional studies are being carried out to determine the feasibility of using this technique for rapid identification of female carriers and colonized neonates. Franciosi and co-workers (8) have indicated that group B strains of type I are most often the cause of the early onset, fulminating form of infection in neonates, whereas types II and III are often found in late onset infection. Although others have failed to find a similar relationship between the type of group B streptococci and the clinical presentation (2), it may be of value to have a rapid means of typing these organisms. The CIE procedure readily distinguished the type Ia and type lb strains from type II and type III strains. Cross-reactions were noted TABLE 3. CIE of 100 clinical isolates of streptococci with group B antisera Lancefield capillary precipitin reaction

No. strains tested

No. positive by CIEB with group antisera

Group B Group A Group C Group G



1 7

0 0 0


TABLE 4. Typing of group B streptococci by CIE Capillary precipitin reaction




a I

1 6 6 7

1 6 0 0

No. positive by CIE Ic C 1

1 6

0 0

0 0




0 0 0 7

VOL. 1, 1975


between the subtypes of type I, but these strains possess common antigens (13). CIE for identification of group B streptococcal isolates offers several advantages over the classic capillary precipitin method. Considerably less technician time is required, since no extraction procedure is necessary. In addition, the results may be available immediately when cerebrospinal fluid or other body fluids are tested and in 5 h when group B streptococcal colonies are identified on culture plates. The classic capillary precipitin test requires 18 to 24 h for growth of the organism in broth, followed by extraction, neutralization, and testing. The apparatus for performing CIE should be available in all hospital laboratories, and antiserum can be obtained from commerical sources at nominal cost. LITERATURE CITED 1. Baker, C. J., and F. F. Barrett. 1973. Transmission of Group B streptococci among parturient women and their neonates. J. Pediatr. 83:919-925. 2. Baker, C. J., F. F. Barrett, R. C. Gordon, and M. D. Yow. 1973. Suppurative meningitis due to streptococci of Lancefield Group B: a study of 33 infants. J. Pediatr. 82:724-729. 3. Barton, L. L., R. D. Feigin, and R Lins. 1973. Group B beta hemolytic streptococcal meningitis in infants.


Pediatrics 82:719-723. 4. Dajani, A. S. 1973. Rapid identification of beta hemolytic streptococci by counterimmunoelectrophoresis. J. Immunol. 110:1702-1705. 5. Edwards, E. A. 1971. Immunologic investigations of meningococcal disease. J. Immunol. 106:314-317. 6. Eickhoff, T. C., J. 0. Klein, A. K. Daly, D. Ingall, and M. Finland. 1964. Neonatal sepsis and other infections due to Group B Beta-hemolytic streptococci. N. Engl. J. Med. 271:1221-1228. 7. Fossieck, B., Jr., R. Craig, and P. Y. Patterson. 1973. Counterimmunoelectrophoresis for rapid diagnosis of meningitis due to Diplococcus pneumoniae. J. Infect. Dis. 127:106-109. 8. Franciosi, R. A., J. 0. Knostman, and R. A. Zimmerman. 1973. Group B streptococcal neonatal and infant infections. J. Pediatr. 82:707-718. 9. Hood, M., A. Janney, and G. Dameron. 1961. Beta hemolytic streptococcus Group B associated with problems of the perinatal period. Am. J. Obstet. Gynecol. 82:809-818. 10. Ingram, D. L., P. Anderson, and D. H. Smith. 1972. Countercurrent-immunoelectrophoresis in the diagnosis of systemic disease caused by Hemophilus influenzae type b. J. Pediatr. 81:1156-1159. 11. Lancefield, R. C. 1933. Serological differentiation of human and other groups of hemolytic streptococci. J. Exp. Med. 57:571-595. 12. Lancefield, R. C. 1934. A serological differentiation of specific types of bovine hemolytic streptococci (group

B). J. Exp. Med. 59:441-458. 13. Wilkinson, H. W., and R. G. Eagon. 1971. Type-specific antigens of group B type Ic streptococci. Infect. Immun. 4:596-604.

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