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Correspondence |
Department of Microbiology, Dr A. L. Mudaliar Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai-600113, Tamil Nadu, India
Correspondence
Thangam Menon
(thangam56{at}gmail.com)
Streptococcus pyogenes or group A streptococcus (GAS) causes a wide spectrum of diseases that include tonsillitis, pyoderma, erysipelas, scarlet fever, necrotizing fasciitis and toxic shock syndrome, as well as non-suppurative complications, such as rheumatic fever and post-streptococcal glomerulonephritis. GAS infections and their sequelae are an especial disease burden in developing countries (Cunningham, 2000).
Epidemiological studies of GAS strains relied historically on M and T serotyping, and more recently on emm gene sequencing, which has become a useful molecular epidemiological tool with which to survey and monitor GAS isolate diversity (Beall et al., 1997). vir typing involves strain discrimination based on the vir regulon, a 4–7 kb pathogenicity locus that encodes structurally related genes of the emm gene family, namely fcrA encoding IgG Fc receptor, emm encoding M protein and enn encoding IgA binding proteins. The vir regulon is flanked by the C5a peptidase encoding gene (scpA) and the regulatory gene virR (Gardiner et al., 1995). Architectural heterogeneity in this region is a consequence of polymorphism exhibited by members of the emm gene family (Gardiner & Sriprakash, 1996). vir typing is the method of choice for studying large numbers of strains in endemic areas by laboratories that do not have the resources for an automated sequencer (Gardiner et al., 1998). The objective of this study was to assess the molecular heterogeneity of GAS isolates in Chennai city using vir typing.
A total of 130 GAS isolates obtained between 2004 and 2005, from children aged 5 to 17 years in Chennai city, were vir typed. They comprised 69 isolates from asymptomatic pharyngeal carriers, 37 isolates from tonsillitis sufferers and 24 isolates from pyoderma sufferers. A total of 59 strains were isolated from males and 71 from females. Of these isolates, 45 were collected during the monsoon season, 58 during winter and 27 during summer.
DNA was extracted from fresh subcultures of GAS in 5 % blood agar by alkali lysis, and the vir regulon amplified by the method described by Hartas et al. (1998), which was based on the method described by Gardiner et al. (1995). The 50 µl PCR mixture contained 5 µl template DNA, 0.4 µM VUF and SBR primers, 5 µl 10x Pfu buffer, 200 µM dNTPs and 0.2 µl 8 : 1 mixture of Taq and Pfu thermostable polymerases. The PCR reaction cycle used was 1 cycle at 95 °C for 1 min, and 30 cycles at 95 °C for 15 s, 60 °C for 2 min and 68 °C for 6 min. The PCR product was run in 0.8 % agarose at 100 V to confirm a 3.5–7.5 kb product, and was digested at 37 °C for 1 h with 2 U HaeIII. The restricted products were run in 1.5 % agarose at 100 V. The patterns obtained were compared for similarity.
A total of 32 vir type (VT) patterns were obtained from the 130 strains, accounting for 24.6 % diversity among them. Of the 130 vir-typed strains, the most common VT was VT1 (23.8 %, 31/130), which was the most common genotype among carriers, tonsillitis and pyoderma patients, and the most common genotype in all seasons and among both sexes (Table 1
). There was only a single representative strain of each of VT20 to VT32.
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A study in North India showed that 40 GAS strains could be differentiated into 16 VTs (Sagar et al., 2004) and another study identified more than 20 profiles among 30 strains (Dey et al., 2005). The present study is the first study on VT diversity of GAS isolates in South India. Our study indicates that the strains of GAS circulating among school children in Chennai belong to several different VTs. Since vir regulon polymorphism is reflective of the heterogeneity of the emm and emm-like genes (Dey et al., 2005), a good deal of M-type diversity could also be expected. In the absence of an emm gene sequencing facility, our method proved to be a good epidemiological tool for identifying genotypes of S. pyogenes. Most studies on GAS genotypes are based on the analysis of isolates exclusively from GAS infections. Our study included isolates from asymptomatic carriers and patients with non-invasive infections, and would thus be indicative of strains circulating in the community. The data may thus contribute to the rational design of a vaccine against genotypes relevant to this region.
ACKNOWLEDGEMENTS
The authors are grateful to the Council of Scientific and Industrial Research, India, for funding this study.
REFERENCES
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Cunningham, M. W. (2000). Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 13, 470–511.
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Hartas, J., Hibble, M. & Sriprakash, K. S. (1998). Simplification of a locus-specific DNA typing method (Vir typing) for Streptococcus pyogenes. J Clin Microbiol 36, 1428–1429.
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