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This study was designed to identify the predominant serotypes of group A streptococci (GAS) responsible for rheumatic fever (RF) and rheumatic heart disease (RHD) in India. RF and RHD sera were screened for antibodies to M proteins retrospectively against known rheumatogenic M types and M types isolated from patients with acute RF. All GAS strains isolated from four patients with acute RF in a short outbreak of RF, were identical-serum opacity factor (SOF) negative, T-pattern 3/13 B3264 and M-non-typable. Because of this, M protein was isolated from only one of these four M-non-typable strain (S-399). This was done by limited pepsin digestion and purification by ion-exchange chromatography on DEAE-Sephadex, followed by gel filtration. Purified M protein was found to be homologous on SDS-PAGE (mol.wt 20 kDa), immunogenic in rabbits and to retain its antigenic structure. This purified M protein from an M-non-typable strain (pep-MNT) was used as an antigen to screen RF and RHD sera retrospectively by ELISA for the prevalence of the M-non-typable GAS strain. The prevalence of the M-non-typable strain was compared with that of the known rheumatogenic M types. Results suggest that the M-non-typable strain could be a provisional new rheumatogenic M type in India and could be a candidate for a multivalent M-protein vaccine to control RF and RHD.