Seven-year surveillance of north american pediatric group a streptococcal pharyngitis isolates.

BACKGROUND: Pharyngeal group A streptococcal (GAS) emm type surveillance enhances understanding of the epidemiology of pharyngitis and invasive GAS disease and formulation of multivalent type-specific vaccines. In addition, such surveillance provides pre-GAS vaccine baseline data. We assessed geographic and temporal trends in GAS emm-type distribution among pediatric pharyngeal isolates collected systematically in the United States and Canada from 2000 to 2007. METHODS: We collected approximately 100 acute GAS pharyngitis isolates from each of 13 widely scattered sites (10 in the United States and 3 in Canada) annually for 7 seasons (2000-2007) from 3- to 18-year-old children. We assessed emm type and subtype by DNA sequencing and analyzed temporal and geographic trends. RESULTS: A total of 7040 US and 1434 Canadian GAS isolates were studied. The 6 most prevalent emm types (in descending order) were 1, 12, 28, 4, 3, and 2 in the United States and 12, 1, 28, 4, 3, 2, and 77 in Canada, constituting 70%-71% of isolates in each country; 10 emm types constituted 87%-89% total. Fifty-six emm types were identified in the United States, including 8 new types, and 33 types in Canada. Although a few types predominated nationally, marked variability among individual sites and at individual sites from year to year was observed. US-Canadian differences in type distribution were apparent. Twenty percent of isolates represented emm subtypes that differed slightly from reference types; 110 new subtypes were identified. An experimental 26-valent M protein vaccine covers 85% of pharyngitis isolates. CONCLUSIONS: Although overall US and Canadian emm type distribution was consistent and relatively few types dominated nationally, striking intersite and temporal variations within individual sites in prevalent emm types of GAS occurred. These results have important implications for the development and formulation of type-specific GAS vaccines.

Genetic relationships deduced from emm and multilocus sequence typing of invasive Streptococcus dysgalactiae subsp. equisimilis and S. canis recovered from isolates collected in the United States.

Beta-hemolytic group C and G streptococci cause a considerable invasive disease burden and sometimes cause disease outbreaks. Little is known about the critical epidemiologic parameter of genetic relatedness between isolates. We determined the emm types of 334 Streptococcus dysgalactiae subsp. equisimilis isolates, and attempted emm typing of 5 Streptococcus canis isolates from a recent population-based surveillance for invasive isolates. Thirty-four emm types were observed, including one from S. canis. We formulated multilocus sequence typing (MLST) primers with six of the seven loci corresponding to the Streptococcus pyogenes MLST scheme. We performed MLST with 65 of the 334 surveillance isolates (61 S. dysgalactiae subsp. equisimilis isolates, 4 S. canis isolates) to represent each emm type identified, including 2 to 3 isolates for each of the 25 redundantly represented emm types. Forty-one MLST sequence types (STs) were observed. Isolates within 16 redundantly represented S. dysgalactiae subsp. equisimilis emm types shared identical or nearly identical STs, demonstrating concordance between the emm type and genetic relatedness. However, seven STs were each represented by two to four different emm types, and 7 of the 10 S. dysgalactiae subsp. equisimilis eBURST groups represented up to six different emm types. Thus, S. dysgalactiae subsp. equisimilis isolates were similar to S. pyogenes isolates, in that strains of the same emm type were often highly related, but they differed from S. pyogenes, in that S. dysgalactiae subsp. equisimilis strains with identical or closely similar STs often exhibited multiple unrelated emm types. The phylogenetic relationships between S. dysgalactiae subsp. equisimilis and S. pyogenes alleles revealed a history of interspecies recombination, with either species often serving as genetic donors. The four S. canis isolates shared highly homologous alleles but were unrelated clones without evidence of past recombination with S. dysgalactiae subsp. equisimilis or S. pyogenes.

Genetically diverse group A streptococci from children in far-western Nepal share high genetic relatedness with isolates from other countries.

The genetic diversity of group A streptococci (GAS) throughout much of the world has not been adequately explored. To assess genetic variation among GAS in western Nepal, 120 noninvasive GAS, collected from eight different villages, were genetically characterized using emm typing, sof sequencing, and multilocus sequence typing (MLST). A high level of genetic diversity was observed among these isolates, with 51 genotypes based upon 51 multilocus sequence types (STs), 45 emm sequence types, and 28 sof sequence types. On the basis of shared ST-emm and sof-emm associations, 40 of the 51 genotypes were identical or highly related to genotypes characterized from locations outside of Nepal, even though most of the emm sequence and clonal types are rare among GAS within the United States. When analyzing all known STs highly related to Nepal STs, only one example of similar STs shared between a sof PCR-positive strain and a sof PCR-negative strain was observed. Since previous data indicate free exchange of MLST loci between sof-positive and sof-negative strains, there is possibly selection against the expansion of subclones resulting from horizontal transfers of sof or emm genes between sof-positive and sof-negative strains. All 45 emm types encountered in Nepal have also been documented from other countries. These data, together with data encompassing the past decade of emm type surveillance, support the possibility that most existing GAS emm types have been discovered. Similarly, since most (40/51) strain types were highly related to strains found elsewhere, it is likely that a major fraction of the existing GAS clonal complexes have been discovered.

Group A streptococcal pharyngitis serotype surveillance in North America, 2000-2002.

Geographic and interseasonal heterogeneity of pharyngeal group A streptococcal (GAS) genotypes (emm types) is poorly characterized. We evaluated emm type and subtype distribution among pediatric pharyngitis isolates obtained from 9 sites in the United States during 2000-2001 (year 1) and from 10 sites in the United States and 1 site in Canada during 2001-2002 (year 2). The 7 predominant types were the same in both years, although their order changed. emm 12, 1, and 28 accounted for 49.2% of year 1 isolates, and emm 1, 12, and 4 accounted for 47.1% of year 2 isolates; 6 types accounted for 72.1% in year 1 and 69.4% in year 2. From year 1 to year 2, the proportions of emm 12 and 28 decreased and emm 1 and 6 increased. Striking intersite and interseasonal variations in the distribution of predominant emm types were observed. We conclude that the most-predominant GAS genotypes were similar for each year despite fluctuations, that intersite and intrasite variations in the distribution of emm types were apparent, and that emm type surveillance is needed as M protein vaccine development proceeds.

Array of M protein gene subtypes in 1064 recent invasive group A streptococcus isolates recovered from the active bacterial core surveillance.

Using sequence analysis to detect variation within the hypervariable M protein N terminus, we found 41 emm types encompassing 81 subtypes, among 1064 consecutive invasive group A streptococcus isolates from a recent multistate, population-based surveillance. Seventeen of the 30 emm types represented by multiple isolates displayed multiple subtypes. Most subtypes differed from reference strain emm sequences as a result of single base substitutions or other alterations likely to be stably inherited. The Centers for Disease Control and Prevention database (available at: http://www.cdc.gov/ncidod/biotech/strep/strepblast.htm) currently contains 225 distinct emm types encompassing 450 subtypes. Although this subtyping scheme increases specificity, limited variation within individual types favors introduction of M protein type-specific vaccines.

Clonal distribution of invasive pneumococcal isolates from children and selected adults in the United States prior to 7-valent conjugate vaccine introduction.

Theseven-valent pneumococcal conjugated polysaccharide vaccine PC7V was licensed for use among children in 2000. Since 90 serotypes of pneumococci exist, an increase in nonvaccine serotypes could occur through immune selection for capsular type switching. Eleven hundred sixty-eight invasive isolates (24 serotypes), recovered primarily from pediatric patients (855 isolates = 73%) and 22 reference strains of known multilocus sequence types (STs) were subjected to macrorestriction profiling (pulsed-field gel electrophoresis [PFGE]). The correlation of 187 ST results (including 49 newly discovered STs) with the PFGE data assigned 1,042 (89.2%) study isolates to 46 defined clonal complexes or genetic lineages based on related multilocus STs (BURST). Seventeen clonal complexes were represented by 2 to 10 related allelic profiles (STs), while 33 lineages (including reference strains) consisted of single STs with 4 or fewer allelic identities to other STs found in the study. Expansion of the BURST analysis to a global analysis of all known pneumococcal STs (as of 27 November 2002) reduced the number of single ST lineages from 33 to 8, and the number of multi-ST clonal complexes was reduced from 17 to 13. In this work, we established the basic genetic structure within individual serotypes prior to PC7V use. The resultant database will be useful for detecting potential selective effects of this vaccine in postvaccine surveillance.

Molecular genetic analysis of a group A Streptococcus operon encoding serum opacity factor and a novel fibronectin-binding protein, SfbX.

The group A Streptococcus (GAS) sof gene encodes the serum opacity factor protein, which is capable of opacifying mammalian sera and binding at least two host proteins, fibronectin and fibrinogen. The sof gene exists in approximately 50% of clinical isolates, and there is a classical association of so-called nephritogenic strains with the opacity factor-positive phenotype. In both a type emm49 strain and a type emm12 strain, the sequences upstream of the 5' end of sof and downstream of the putative terminator were determined to be nearly identical to a region in the M type 1 genome approximately 10 kb upstream of the emm1 gene. This close genetic linkage is likely reflected in the strict correlation of opacity factor phenotype with specific emm genotypes. A new fibronectin-binding protein gene, sfbX, was discovered immediately downstream of sof in emm12 and emm49 strains and in several other sof-positive strains. The sof and sfbX genes were found to be expressed on the same transcription unit, which was correlated with the putative promoter and rho-independant terminator sequences that flank these two genes. The sfbX genes from different emm types are predicted to encode approximately 650-residue surface-bound proteins sharing 89 to 92% sequence identity. SfbX residues approximately 1 to 480 are not highly similar to those of other known proteins, with the closest match being the Staphylococcus aureus coagulase protein. The remaining portions of these proteins (residues 481 to 650) contain four putative fibronectin-binding repeats highly similar to those of other streptococcal fibronectin-binding proteins and a potential LP(X)SG cell wall anchor motif. Targeted in-frame allelic-exchange mutagenesis, complementation, and heterologous-expression studies found that serum opacification is encoded by sof alone and that sfbX encodes a fibronectin-binding function. A recombinant SfbX protein was found to bind immobilized fibronectin and to partially inhibit GAS adherence to fibronectin. The sfbX gene was found to be present only in sof-positive strains, and together these genes could influence the spectrum of tissues colonized by sof-positive GAS.

M protein gene type distribution among group A streptococcal clinical isolates recovered in Mexico City, Mexico, from 1991 to 2000, and Durango, Mexico, from 1998 to 1999: overlap with type distribution within the United States.

To examine the type distribution of pathogenic group A streptococcal (GAS) strains in Mexico, we determined the emm types of 423 GAS isolates collected from ill patients residing in Mexico (Durango or Mexico City). These included 282 throat isolates and 107 isolates from normally sterile sites. Of the other isolates, 38 were recovered from other miscellaneous infections. A total of 31 different emm types were found, revealing a broad overlap between commonly occurring emm types in Mexico and the United States. The information obtained in this study is consistent with the possibility that multivalent, M type-specific vaccines prepared for GAS strain distribution within the United States could theoretically protect against the majority of GAS strains causing disease in the two cities surveyed in Mexico.