Rapid tube CAMP test for identification of Streptococcus agalactiae (Lancefield group B).

A rapid CAMP test for the presumptive identification of Streptococcus agalactiae (Lancefield group B) is described. Sheep erythrocytes, sensitized by staphylococcal beta-lysin and suspended in phosphate-buffered saline, were used to determine the lytic capacity of the neutralized supernatant fluids of 4-h broth cultures of streptococci being tested. A total of 96.2% of 130 group B streptococci gave positive CAMP tests, that is, lysis of the sheep erythrocytes after 10 min of exposure of streptococcal supernatants, whereas none of 381 non-group B streptococci tested produced any lysis. The test described provides presumptive identification of group B streptococci within 4 h and eliminates problems of intermediate reactions so that a positive test is indicative only of CAMP factor production.     

Typing ofStreptococcus agalactiae (Lancefield group B)

A two-stage typing scheme in routine use in this laboratory is described. The strains of group B streptococci (GBS) are first serotyped and then, if necessary, phage-typing is performed. Serotyping lacks discrimination because almost 40% of strains handled carry either the Ia/c or III/R antigens. On the other hand, the typability rate with phages is less than 80%. The evidence from the combined scheme is consistent with the view that GBS infections occurring within the first five days of life are, with few exceptions, due to acquisition of the mother's flora. After this time the infecting strain may come from other sources. Human and bovine strains of GBS belong predominantly to two recognisably different populations that can be distinguished by antigenic and biochemical differences. There are no patterns of lysis by phages characteristic of either human or bovine strains.     

Possible virulence marker forStreptococcus agalactiae (Lancefield group B)

Mouse passage of a stock strain of each of the serotypes Ia, Ib, Ia/c, II and III ofStreptococcus agalactiae was followed by increased virulence. The change was associated with an increased content of sialic acid and also with the appearance of an antigen common to all passaged strains. This antigen was subsequently detected in all but one of 12 strains isolated from infected babies or diabetic adults, but in none of 12 organisms recovered from carriers.     

Lancefield grouping and smell of caramel for presumptive identification and assessment of pathogenicity in the Streptococcus milleri group.

AIM: To evaluate Lancefield grouping and caramel smell for presumptive identification of the Streptococcus milleri group, and to find whether Lancefield group, species, or protein profile correlated with virulence or infection site. METHODS: Prospective studies were made of 100 consecutive streptococcal isolates in blood cultures or pus from 100 patients in whom the severity of infection was categorised as serious, moderate, or not significant. The usefulness of Lancefield group and the caramel smell for presumptive identification was examined, and the relation of the S milleri species, Lancefield group, and SDS-PAGE protein analysis to severity of infection and infection site was investigated. Lower respiratory tract and genital tract specimens, strict anaerobes, group D streptococci, and strains identified as Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcus agalactiae were excluded. RESULTS: Most streptococci occurring in pure or significant growth density were S milleri group (87/100; 87%, 95% confidence interval 0.81-0.93). Of these, 89.7% (78/87; 0.84-0.96) were associated with infection. Lancefield group F antigen predominated (41/87; 47.1%, 0.38-0.56). Lancefield group F alone or accompanied by the caramel smell had a specificity of 100%, but a sensitivity of only 47.3% for group F alone, and 19.5% for group F accompanied by the caramel smell. There was no significant association between species, Lancefield group, and severity of infection, site of infection, or pathogenicity. SDS-PAGE analysis failed to discriminate between strains. CONCLUSIONS: Neither species nor Lancefield antigen was related to the site of infection. The presence of Lancefield group F antigen alone or accompanied by a caramel smell was a useful indicator for the S milleri group when present, but was too insensitive to use as a screening test. Most streptococci occurring in pure culture or in significant growth density were of clinical importance. Such organisms should be identified to species level to detect the S milleri group.     

Use of a serotype-specific DNA microarray for identification of group B Streptococcus (Streptococcus agalactiae)

Group B Streptococcus (GBS; Streptococcus agalactiae) is an important cause of sepsis and meningitis. Nine GBS serotypes, based on capsular polysaccharide (CPS) antigens, have been described. Their distribution varies worldwide and needs to be monitored to understand the epidemiology of GBS disease and inform the development of vaccines. In this study, we sequenced cpsH of GBS serotype II (cpsHII) and compared it with that of the other eight serotypes to identify serotype-specific regions. We then developed a DNA microarray based on the cpsH gene and used it to test 88 GBS isolates-9 serotype reference strains and 79 clinical isolates-and 7 other bacterial and fungal species which are commonly present in the vagina flora. The microarray was shown to be specific and reproducible. This is the first report of a microarray which can identify the nine GBS serotypes. The use of a microarray has advantages over traditional serotyping methods and will be of practical value in both reference and diagnostic laboratories.     

Toxicity of group B Streptococcus agalactiae in adult rats.

Several strains of group B Streptococcus agalactiae were found to be lethal for young adult rats. When bacteria were heat killed and then injected intraperitoneally into rats, rapid death (14 to 18 h) of the rats occurred, characterized by labored breathing, hemolyzed serum, hemoglobinuria, and subungual hemorrhages. Sections of tissues from these rats failed to reveal the cause of death. Rats injected with toxic or nontoxic strains of group B S. agalactiae had reduced numbers of circulating leukocytes and low serum C3 levels in comparison with those in control rats. The toxic strains of group B S. agalactiae induced dramatic decreases in platelet numbers, and in plasma fibrinogen levels as well, suggesting that the toxicity was due to disruption of the coagulation system. Rapid death in the absence of infection suggests that group B S. agalactiae may have a cell-associated toxin that induces these changes. Such a toxin may be a contributory factor in the high mortality rate associated with group B streptococcal infections of the human neonate.     

Clinical laboratory evaluation of a reverse CAMP test for presumptive identification of Clostridium perfringens.

Ninety-six percent of Clostridium perfringens isolates from clinical specimens were reverse CAMP test positive, whereas several other Clostridium species tested were reverse CAMP test negative. C. perfringens was detected by direct inoculation of clinical specimens to reverse CAMP plates, and the reverse CAMP procedure provided reliable presumptive identification of this organism.     

CAMP-disk test for presumptive identification of group B streptococci.

Standaridizing test results for rheumatoid factor by comparing results obtained for an unknown with results obtained for a serum reference preparation decreased variance between laboratories, as measured in the Center for Disease Control proficiency testing program, by 77%. The amount of improvement was also estimated by the type of test and by the manufacturer's product. Standardization resulted in an increase in the number of reported results that were within a twofold dilution of the median value. The percentage increased from 50.3 to 93.7% for the slide tests and from 78.1 to 91.2% fro the tube tests. Decrease in variance by manufacturer's product ranged from 94 to 27%. The study demonstrated that adopting a reference serum standard could substantially improve the comparability of rheumatoid factor test results and that proficiency testing programs can be used to estimate improvement which could be expected as a result of standardization.     

Detection of diacetyl (caramel odor) in presumptive identification of the "Streptococcus milleri" group.

The caramel odor associated with the "Streptococcus milleri" group was shown to be attributable to the formation of the metabolite diacetyl. Levels of diacetyl in the 22- to 200-mg/liter range were produced by 68 strains of the "S. milleri" group; apart from one strain of Streptococcus mutans, all 92 other strains of streptococci belonging to 12 species produced < 13 mg of diacetyl per liter. Quantitation of diacetyl levels from cultures of streptococci is suggested as a rapid presumptive test for the "S. milleri" group.     

Evaluation of spot CAMP test for identification of group B streptococci.

The CAMP (Christie-Atkins-Munch-Petersen) test is commonly used for the presumptive identification of Streptococcus agalactiae (Lancefield group B). Using 350 clinical isolates of beta-hemolytic streptococci, we compared a 30-min spot CAMP test with the standard overnight CAMP test and the Lancefield precipitin test. We found 99% agreement among all three tests for all streptococci tested. The spot CAMP test is a rapid, inexpensive, and accurate method for identifying group B streptococci.     

Liquid medium for rapid presumptive identification of group B streptococci.

A suitable test was developed for distinguishing group B streptococci from other beta-hemolytic streptococci during growth in liquid medium. One hundred and sixty of 161 human group B strains tested yielded positive reactions within a 5 h incubation. The dye medium tested is a reliable substitute for more expensive serological procedures.     

Further studies on the reliability of the bacitracin inhibition test for the presumptive identification of Lancefield group A streptococci.

The reliability of the bacitracin inhibition test to differentiate between 125 Lancefield group A and 122 non-group A beta-haemolytic streptococci was studied. Bacitracin discs containing 0-02, 0-04, or 0-1 international units and the conditions recommended by both the Association of Clinical Pathologists and the Federal Drug Administration for routine sensitivity testing were used. The results suggest that the presence or absence of a zone of inhibition around a 0-04 unit disc can be used routinely for the presumptive identification of group A streptococci and that a specific zone size can be used for reading the test only with a 0-1 unit disc.     

International external quality assurance for laboratory identification and typing of Streptococcus agalactiae (Group B streptococci)

We report the results from the first international multicenter external quality assessment (EQA) studies for molecular and serological typing of group B streptococcus (GBS) strains as part of DEVANI (Design of a Vaccine against Neonatal Infections), a pan-European program. A questionnaire-based surveillance was undertaken among eight laboratories participating in DEVANI and six laboratories not participating in DEVANI from 13 countries in order to assess their current microbiological procedures for GBS screening, diagnosis, and typing. GBS strains from three EQA distributions were characterized using molecular and serological methods based on GBS capsular polysaccharide typing. Participants were asked to test the first distribution using their current serotyping and genotyping methods. The Strep-B-Latex agglutination method was the most widely used method, with a typeability value of >90%. A multiplex PCR assay for GBS capsular gene typing was also used by 2 of 14 centers, which achieved a typeability value of 93%; this assay detected only 9 of 10 GBS capsular polysaccharide genes. From the second and third EQA studies, standardized protocols were prepared for serological and molecular typing of GBS strains based on the Strep-B-Latex agglutination method and a novel multiplex PCR assay that detected all 10 GBS capsular types (Ia to IX). These standardized protocols are being used by many European laboratories, and as the use of these methods increases, it is imperative to continuously improve and assess laboratory performance and offer training to any laboratories that have technical difficulties.     

Typing of human isolates of Streptococcus agalactiae (group B streptococcus,GBS) strains from Zimbabwe

Serotyping and genotyping are important tools in epidemiological studies of group B streptococcal (GBS) infections, which are important diseases in man, particularly in newborns. In the present study, 241 GBS isolates from Zimbabwe, comprising 124 carrier isolates from pregnant women and 117 isolates from patients hospitalised for various diseases, were serotyped. Antibodies specific for the capsular polysaccharide antigens (CPAs) Ia, Ib and II-V and antibodies specific for the surface-localised proteins, c(alpha), c(beta), R1, R3 and R4 were used for serotyping. Strains of the CPA types Ia (17%), III (47.7%) and V (23.2%) predominated. Of the various protein antigens, c(alpha) and R4 were expressed with highest frequency, c(alpha) by 100% of the CPA type Ia strains and R4 by 92% of the CPA type III strains. The R3 protein occurred frequently (24%), especially in type V strains (84%). A total of 25 serovariants was detected in the strain collection with the variants Ia/c(alpha) (16%), III/R4 (43.5%) and V/c(alpha), R3 (14.1%) occurring with the highest frequency. Serotype and subtype distribution of the carrier isolates were essentially similar to those of the disease-associated isolates. Genomic heterogeneity was demonstrated by pulsed-field gel electrophoresis of type III/R4 and type V/c(alpha), R3 isolates, but to a much lesser extent than recorded with Norwegian strains. These results demonstrate that many variants of GBS occur in the Zimbabwean population. The data obtained may assist in the formulation of a possible future GBS vaccine for Zimbabwe and perhaps for other African countries.     

Bile medium for rapid presumptive identification of group B streptococci

A bile medium was developed for rapid presumptive identification of beta hemolytic group B streptococci. Of 131 clinical isolates of group B streptococci, 125 (95.4%) yielded positive reaction within 6 h incubation. No false positive reaction was found in 134 clinical isolates of groups A, C, F, and G beta-hemolytic streptococci. The sensitivities of the bile medium, rapid hippurate hydrolysis and modified LAL-1 medium were 95.4, 98.5 and 93.1%, respectively. All three media showed 100% specificity. Therefore, the bile medium provides as an additional medium for rapid presumptive identification of group B streptococci.     

Streptococcus equi subspecies equi (Lancefield group C) meningitis in a child

We present a case of Streptococcus equi subsp. equi meningitis in a young boy. This case represents the first report in the literature of meningitis caused by this organism, as far as we know.     

Isotype antibody response in cows to Streptococcus agalactiae group B polysaccharide-ovalbumin conjugate.

Adult dairy cows were immunized with group B antigen (GBA) of Streptococcus agalactiae or GBA coupled to ovalbumin, both emulsified in incomplete Freund adjuvant, and their sera were examined by an enzyme-linked immunosorbent assay measuring bovine immunoglobulin isotypes (immunoglobulin G1 [IgG1], IgG2, and IgM) specific for GBA. All of the cows possessed naturally acquired antibodies against GBA, which implied that primary antibody responses could not be studied. At the highest dose tested (200 micrograms), free GBA elicited a slight increase in antibody titers only in the IgM isotype, to which most of the naturally acquired antibodies to GBA belonged. A second administration of antigen was not more effective. The conjugate was able to induce a strong humoral response against GBA, particularly in the IgG1 and IgG2 subisotypes, and a second injection of the conjugate induced a doubling of the peak antibody titers. Therefore, conjugation of GBA to a protein carrier markedly improved the antibody response, which showed the main characteristics of T-cell dependency. The opsonic activity of serum against an unencapsulated strain of S. agalactiae was reinforced by the immunization with the conjugate.     

Antigenic determinant of the Lancefield group H antigen of Streptococcus sanguis.

Previous studies indicated that the teichoic acid isolated from strains of Streptococcus sanguis was group specific and defined the Lancefield group H streptococci. To determine the specific antigenic determinants, the antigen was extracted from a group H streptococcus (ATCC 903) by the phenol-water method and purified by column chromatography. The isolated antigen had a glycerol/phosphate/glucose molar ratio of 1:0.9:0.3; the lipid concentration was 7.6% of its dry weight. No nucleic acids were detected, and amino acids constituted approximately 2% of the dry weight. The minimum concentration of antigen required to sensitize erythrocytes for hemagglutination with a 1:1,000 dilution of either group H antiserum or antiteichoic acid serum was 0.02 microgram/ml. Hemagglutination inhibition studies suggested that the major antigenic determinant consisted of an alpha-glucose linked to the glycerol phosphate backbone.