International External Quality Assurance for Laboratory Identification and Typing of Streptococcus agalactiae (Group B Streptococci)

Abstract

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.

Genotyping of Clinical Streptococcus agalactiae Strains Based on Molecular Serotype of Capsular (cps) Gene Cluster Sequences Using Polymerase Chain Reaction

“…Molecular Serotyping identification methods are attractive due to several advantages including being specific, their high discriminatory power, clear results, high reproducibility, simplicity and fast to perform, and great availability of equipment and required materials. Advanced molecular methods have capability of differentiating strains to many variable types (4,15,30).…”

Capsular Gene Typing of Streptococcus agalactiae Compared to Serotyping by Latex Agglutination

“…However, the results identify a problem with the serotype V and IX latex reagents (Statens Serum Institute) used for the agglutination tests (Table 1). Moreover, the accuracy of the results is highly dependent on experience (10), and the latex agglutination assay is less sensitive than the flow cytometric method as demonstrated by our observation that approximately one-half of the strains scored as nontypeable in this assay were found to express type-specific polysaccharides when examined by the flow cytometric method. The exact proportion of unselected isolates that would present this problem in clinical microbiology is not clear, as the strain collection examined in this study was enriched with strains that were difficult to type in other laboratories.…”

“…Serological methods have limitations, as they may fail to type an isolate due to lack of or low expression of capsular polysaccharide under the experimental conditions. Furthermore, these methods are highly dependent on the quality of the antibodies used and on the experience of the laboratory (10). Therefore, capsular gene typing is an attractive alternative.…”

“…A heavy suspension of the test organism harvested from the blood agar plate was prepared in 250 l phosphate-buffered saline (PBS), pH 7.4. For each of the 10 serotypes, a 20-l aliquot of the bacterial suspension was applied to a Wellcogen disposable reaction card and mixed with 1 l of latex suspension (reagents Ia, Ib, and II to IX; Strep-B-Latex kit; Statens Serum Institut, Copenhagen, Denmark) as re-cently described (10). The reaction card was rotated slowly and observed for agglutination, and a positive reaction was scored when clear-cut agglutination appeared within 30 s. Notably, false-positive reactions may occur if the reaction time exceeds 30 s. Serotyping by latex agglutination and the capsular gene typing described below were performed in the same laboratory at Aarhus University.…”

Identification of Group B Streptococcus Capsule Type by Use of a Dual Phenotypic/Genotypic Assay

“…The sensitivity of the serological assays used to detect GBS capsule polysaccharide can vary. These methods depend on the quality of the antibodies, the technical experience of the operator, and the expression of a detectable amount of capsule (53). A study by Kilian and coworkers (54) identified limitations associated with GBS latex agglutination serotyping.…”