Novel PCR-Restriction Fragment Length Polymorphism Method for Determining Serotypes or Serogroups of Streptococcus pneumoniae Isolates

Abstract

Serotyping Streptococcus pneumoniae is a technique generally confined to reference laboratories, as purchasing pneumococcal antisera is a huge investment. Many attempts have been made to modify serological agglutination techniques to make them more accessible, and more recently developments in serotyping have focused on molecular techniques. This paper describes a PCR assay which amplifies the entire capsulation locus between dexB and aliA. Amplicons are digested to produce serotype-specific patterns. We have shown, using 81 epidemiologically unrelated strains representing 46 different serotypes, that the patterns correlate with a 90 to 100% similarity range for the same serotype or serogroup. Prospective testing of 73 isolates of unknown serotype confirmed reliable serotype attribution, and serotype profiles are reproducible on repeated testing. Once our database contains all 90 serotypes, this technique should be fully portable, cost-effective, and useful in any laboratory with sufficient molecular experience.The species Streptococcus pneumoniae possesses more than 90 serotypes defined by their polysaccharide capsule (1). Immunologically similar serotypes, such as 19F, 19A, 19B, and 19C, are grouped together in serogroups. The immune response to capsular polysaccharide is critical for recovery from infection (17), and the first effective treatment for pneumococcal infection, serum therapy, depended on identifying the infecting serotype rapidly so that type-specific horse serum could be administered (9). The capsule is the focus for the development of an effective vaccine, initially with the 23-valent polysaccharide and more recently the protein-polysaccharide conjugate preparations that are undergoing clinical evaluation and have entered clinical use (1, 35). Some reports on the implementation of the conjugate vaccine in different communities show that there is an increase in the carriage of serotypes that are not included in the vaccine (6, 7). Additionally, serotype surveillance in developing countries and special populations, such as Aboriginal Australians, suggests that vaccine serotype coverage may be lower than for the United States and Europe, from which surveillance data were considered in vaccine formulations (2, 4, 25). Thus, there is a continuing need to study the epidemiology of S. pneumoniae as defined by capsular polysaccharide in monitoring the effect of conjugate vaccines and to aide in the development of new vaccine formulations for developing countries and special populations (12,13,16).Effective serotyping depends on a full set of group-and type-specific antisera prepared by the Statens Serum Institut (14), an investment that is beyond the resources of many laboratories. Therefore, most laboratories confine typing to the serotypes and serogroups found in the 23-valent vaccine, which represent most of the invasive types found in industrialized countries (22). As prevailing serotypes change, a wider selection of sera will be required to cover common types, thus increasing the cost. In additio...

Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy

“…Existing methods targeting cps employing multiple restriction enzymes and/or a long PCR fragment, making amplification difficult and inconsistent, have been described (6,38). A sequence-based typing system targeting the regulatory region of cps had been proposed (34,35), but a low resolution was seen between cross-reactive serotypes, in addition to a lack of reproducibility in other serotypes.…”

“…Pneumococci with known serotypes and multilocus sequence types (STs) from upper respiratory tract samples (URT) were isolated from a cohort of asymptomatic children under the age of six in Tanzania (6,17,42). In addition, laboratory pneumococcal reference strains with known serotypes (some with known STs) from GR MICRO, Health Protection Agency (HPA) laboratories in the United Kingdom (Colindale and Glasgow), and the American Type Culture Collection (ATCC) were included.…”

Sequential Multiplex PCR Approach for Determining Capsular Serotypes of Streptococcus pneumoniae Isolates

“…Though these molecular approaches to serotyping appear promising, the existing systems identify a limited number of serotypes (3,13,14,24) and need to be expanded to establish a more comprehensive approach for routine surveillance. Recently, an approach relying upon amplification of the entire cps locus, followed by restriction fragment length polymorphism analysis, was assessed (2). Although this scheme shows promise, we prefer an approach that does not include segments of the cps locus that are highly conserved between serotypes, but instead relies upon upon short sequences specific to individual serotypes or serogroups.…”

Capsular-type prediction by phylogenetic tree of glycosyltransferase gene polymorphism in Streptococcus pneumoniae

“…[17][18][19][20][21][22][23][24][25][26] In this study, we focused on the GT genes due to their role in forming capsular polysaccharides. Each serotype contains various sets of GT genes in the cps locus.…”