Resumo

Ninety-one Streptococcus pneumoniae serotypes have been described, but the conjugated anti-pneumococcal vaccine distributed by the Brazilian public health system covers only the ten most prevalent in this country. The classical pneumococcal serotyping is expensive, error prone due to cross-reactivity between similar capsular antigenic polysaccharides (CPS). We propose a new tool for molecular serotyping of S. pneumoniae based on restriction fragment length polymorphisms of the PCR-amplified cps loci (cps-RFLP) which encodes the enzymes responsible for CPS synthesis. We predicted the cps-RFLP patterns by in silico digesting the cps loci of the 91 serotypes with the endonucleases XhoII, or HinfI. The latter had already been proposed for molecular serotyping of a subset of pneumococcal serotypes. Using our previously published web service Molecular Serotyping Tool (MST – www.cebio.org/mst), restriction patterns can be aligned and their distance calculated as the sum of the penalties for the edit operations that transform one pattern into the other. MST easily distinguished each serotype-specific cps-RFLP pattern from all other in the database. Taking into account the median of the distances between all pairs of cps-RFLP patterns and the number of indistinguishable pairs for each enzyme, MST was more discriminant with XhoII (median= 32.8; indistinguishable pairs= 7) than HinfI (median=8.9; indistinguishable pairs= 24). The rare cps-RFLP patterns pairs that MST could not distinguish when using XhoII correspond to some of the serotypes with reported cross-reactivity to antisera used in classical serotyping. Molecular serotyping can be automated and does not require the use of animals, being a robust alternative to serological typing. We are validating our method comparing the predicted cps-RFLP patterns with those experimentally obtained for the FUNED’s previously serotyped S. pneumoniae collection. This isolates represent the most prevalent serotypes in Minas Gerais (Brazil). Preliminary results show a strong correlation between the experimental and the predicted cps-RFLP patterns. The final database of cps-RFLP patterns will be made available freely through MST, allowing a real time epidemiological surveillance of serotype shifting, a common phenomenon after massive immunization and supporting continuous improvement of pneumococci vaccines.