Genotyping of group A streptococcus by various molecular methods.

ABSTRACT

BACKGROUND & OBJECTIVE: Group A streptococcus (GAS) causes a wide variety of life threatening diseases in developing countries like India. Characterization of GAS is therefore necessary for prevention and control of the disease. Genotypic analysis of GAS is largely lacking from India, therefore an attempt was made to study the genotype distribution of north Indian GAS isolates. METHODS: Sixty clinical isolates of GAS, (52 collected from pharyngitis and 8 from RF/RHD patients) were genotyped by various molecular techniques like restriction enzyme analysis (REA), ribotyping, PCR-ribotyping and random amplification of polymorphic DNA (RAPD). A few isolates were also typed by emm gene sequencing for comparison. RESULTS: REA using Hind III digestion differentiated the isolates into six different patterns. The same isolates were grouped into three ribotypes when analyzed for PCR - ribotyping of 16S- 23S rRNA region. However, RAPD fingerprints generated higher level of discrimination by AP4 and AP5 primers showing 12 rapdemes, followed by AP3, AP2 and API producing 11, 9 and 6 rapdemes respectively. A total of 78 RAPD fragments or rapdemes were generated, of which 48 (62%) were shared and 30 (38%) were unique. These unique RAPD fragments could be used as a genetic marker for identification of GAS. Representative isolates that produced 12 different rapdemes by AP5, on further confirmation by emm typing showed 11 different emm types. INTERPRETATION & CONCLUSION: The finding of our study demonstrated the RAPD profiling to be the most discriminatory for genotyping of group A streptococcus isolates as well as comparable to the most commonly used sophisticated technique of emm typing.