emm typing of invasive T28 group A streptococci,
Tuula Siljander,1Maija Toropainen,1Anna Muotiala,1Nancy P. Hoe,2
James M. Musser3and Jaana Vuopio-Varkila1
1Hospital Bacteria Laboratory, Department of Bacterial and Inflammatory Diseases, National
Public Health Institute, Mannerheimintie 166, FIN-00300, Helsinki, Finland
2Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute
of Allergy and Infectious Diseases, National Institutes of Health, 903 South Fourth Street,
Hamilton, MT 59840, USA
3Center for Molecular and Translational Human Infectious Diseases Research, The Methodist
Hospital Research Institute, 6565 Fannin Street, Houston, TX 77030, USA
Received 20 April 2006
Accepted 4 July 2006
A total of 985 group A streptococcus (GAS) bacteraemia isolates collected in Finland during
1995–2004 were T-serotyped, and of these, 336 isolates of serotype T28 were subjected to
further emm typing. The total number of isolates referred per year showed an increase within the
study period, from 43 in 1995 to 130 in 2004. The annual incidence of invasive GAS (iGAS)
bacteraemia showed a general increase during the study period, from 1?1 to 2?5 per 100000
population. Serotype T28 remained among the most common serotypes, in addition to serotypes
TB3264 and T1. The serotype T28 isolates were found to be distributed across six distinct emm
types: emm28, emm77, emm53 (including subtypes 53.2 and 53.4), emm87, emm2 and
Withinthestudy period, theproportion of T28/emm28isolates becamethemost prominent.During
revealed T28 isolates to be a genetically heterogeneous group harbouring a variety of distinct M
proteins. This study confirms that T serotyping alone is not a sufficient method for epidemiological
surveillance of iGAS.
Streptococcus pyogenes (group A streptococcus, GAS) causes
tract infections to invasive infections, including cellulitis,
bacteraemia, necrotizing fasciitis and toxic-shock syn-
drome. Invasive GAS (iGAS) diseases constitute a major
global burden, resulting in hundreds of thousands of cases
each year, most of which occur in less-developed countries
(Carapetis et al., 2005). Population-based surveillance
changes in the epidemiology of GAS (Lamagni et al., 2005),
and have indicated the importance of type identification for
epidemiological studies (Efstratiou, 2000). The availability
and application of discriminatory typing methods are
essential to these studies.
The classical serological typing schemes for GAS are based
on the variability of surface-exposed proteins, such as the T
protein, the serum opacity factor (SOF) protein and the M
protein (Johnson et al., 1996). The T-agglutination test
introduced in 1965 has provided a useful tool for initial
screening and characterization of GAS when combined with
the SOF reaction result (Moody et al., 1965; Maxted et al.,
1973; Johnson & Kaplan, 1993; Johnson et al., 1996).
However, the value of the serological typing methods is
limited by the specificity and availability of typing sera.
Conventional methods are therefore being replaced or
augmented by molecular methods.
The major GAS virulence factor, streptococcal M protein,
has multiple functions in the pathogenicity of the
bacterium, including its contribution to the antiphagocytic
capacity of the bacterium, thereby promoting its invasive-
ness (Fischetti, 1989). M proteins or M-like proteins have
been identified also in group C and G streptococci (Collins
introduced by Rebecca Lancefield provided the basis of
GAS M typing, and has been further enhanced by the
implementation of molecular approaches directed towards
Abbreviations: CDC, Centers for Disease Control and Prevention; GAS,
group A streptococcus; iGAS, invasive GAS; KTL, National Public
Health Institute of Finland; MLST, multilocus sequence typing; NIDR,
National Infectious Disease Register; NT, non-typable; SOF, serum
46690 Printed in Great Britain1701
Journal of Medical Microbiology (2006), 55, 1701–1706
the M protein emm gene (Lancefield, 1962; Podbielski et al.,
1991; Beall et al., 1996; Saunders et al., 1997; Facklam et al.,
1999). emm typing is based on the determination of
differences in the 59 end of the emm gene encoding the
hypervariable and outward-projecting amino-terminal por-
tion of the M protein (Fischetti, 1989; Beall et al., 1996).
typing has made it the current ‘gold standard’ method
(Efstratiou, 2000), which for the purpose of detecting
clonality of strains is most useful when complemented
by other molecular typing methods, such as ribotyping,
sof sequencing, PFGE, restriction endonuclease analysis
(REA) and multilocus sequence typing (MLST) (Single
& Martin, 1992; Seppala et al., 1994; Beall et al., 2000;
Enright et al., 2001; McGregor et al., 2004; Doktor et al.,
At present, 83 unique M types encoded by unique emm gene
sequences (up to type M93) have been validated (Facklam
et al., 2002; Johnson et al., 2006). Over 110 emm types (up
to type emm124) have also been validated and submitted
to the Streptococcus pyogenes emm sequence database at
the Centers for Disease Control and Prevention (CDC)
In addition, the database currently contains 47 provisional
sequence types (sts) for S. pyogenes (http://www.cdc.gov/
In this study, serotype T28 was the focus of interest because
of its dominance among Finnish iGAS isolates. Serotype
T28/M28 strains are commonly reported among the top five
most common causes of invasive and pharyngitis infections
in several countries, in addition to being predominant
among cases of puerperal sepsis and neonatal GAS infection
(Colman et al., 1993; O’Brien et al., 2002; Tyrrell et al.,2002;
Moses et al., 2003; Shulman et al., 2004; Green et al., 2005b;
Raymond et al., 2005). In order to have a better under-
standing of the epidemiology and clonality of Finnish T28
isolates, emm typing was performed for all GAS isolates
reacting with anti-T28 typing serum from the study period
Surveillance of invasive GAS disease. In Finland, iGAS disease,
defined as GAS isolated from blood and cerebrospinal fluid, has
been notifiable by law since 1995. Clinical microbiology laboratories
report (generally electronically) all invasive S. pyogenes isolations to
the National Infectious Disease Register (NIDR) at the National
Public Health Institute of Finland (KTL) (http://www3.ktl.fi/stat). In
addition, the corresponding GAS isolates are referred to the national
reference laboratory at KTL for confirmation and typing. In this
study, notifications and isolates concerning S. pyogenes blood isola-
tions were included. The isolates were cross-checked to match the
Bacterial identification. The referred isolates were confirmed as S.
pyogenes by beta-haemolysis on blood agar, sensitivity to bacitracin,
and detection of Lancefield group A antigen by latex agglutination
T serotyping. T serotyping was performed using five polyvalent
and 21 monovalent anti-T agglutination sera (1, 2, 3, 4, 5, 6, 8, 9,
11, 12, 13, 14, 18, 22, 23, 25, 27, 28, 44, B3264 and Imp19) (Sevac),
as described elsewhere (Moody et al., 1965).
Human Bacterial Pathogenesis, Rocky Mountain Laboratories (RML)
(most isolates from 1995 to 1999) and at KTL (mainly isolates from
2000 to 2004). emm typing at RML was performed using primers
emm1b and emm2 by previously documented methods (Green et al.,
2005a). At KTL, primers MF1 (forward), 59 ATA AGG AGC ATA AAA
ATG GCT 39, and MR1 (reverse), 59 AGC TTA GTT TTC TTC TTT
GCG 39, were used under the following conditions: initial denaturation
at 95uC for 10 min and 94uC for 3 min, 35 cycles of denaturation at
93uC for 30 s, annealing at 54uC for 30 s and extension at 72uC for
2 min, with a final extension step at 72uC for 10 min. PCR products
were purified with the QIAquick PCR purification kit (Qiagen), as
described by the manufacturer. The emm sequencing reaction was per-
formed with primer MF1 and BigDye chemistry (Applied Biosystems), as
described by the manufacturer, and analysed with an ABI Prism 310
genetic analyser (Applied Biosystems). The sequence data were compared
with the CDC Streptococcus pyogenes emm sequence database (http://
assigned on the basis of ¢95% sequence identity with the exact 150-
base type-specific sequences as specified by the database (http://
emm typing was performed jointly at the Laboratory of
Statistical analysis. Disease rates were calculated using resident
population data as the denominators for the corresponding year.
Data were analysed using the statistical software Intercooled Stata
9.1 for Windows (StataCorp). Categorical data were compared using
the chi square test. Differences were considered significant when
RESULTS AND DISCUSSION
Epidemiology of iGAS disease in Finland
During 1995–2004, 1034 cases of iGAS disease (blood
bacteraemia) were reported in Finland, and from these, 985
corresponding isolates were submitted to the reference
laboratory at KTL (Fig. 1). The annual incidence of iGAS
bacteraemia showed a general increase, from 1?1 to 2?5 per
100000 population during the period 1995–2004, peaking
submissions also increased threefold, from 43 to 130. The
reasons for this increase are currently undetermined. The
overall coverage of the notification system and sending of
isolates to KTL in Finland is good and has remained stable
over this period. On average, we received an isolate for
typing in 95% of the notified cases. The disease rates of GAS
bacteraemia in Europe have recently been reported to be
between 1?7 and 3?95 per 100000 population in the early
2000s, varying considerably by country but showing a
general increasing trend over the past two decades in most
countries (Lamagni et al., 2005). The Finnish trend follows
this pattern. In the USA, the disease rate of iGAS (including
bacteraemia) has been estimated to be around 3?5 per
100000 population from the late 1990s onwards (http://
et al., 2002).
1702Journal of Medical Microbiology 55
T. Siljander and others
The 985 iGASbloodcultureisolates from 1995to 2004 were
found to be distributed across a total of 41 different T
serotypes. The different serotypes obtained agglutinated
either with a single serum or with multiple sera, in which
case all the positive reactions were listed and formed the
combination serotype name. The dominant serotypes
throughout the period were T28, TB3264, T1, T8 and
T12, all agglutinating with a single serum (Fig. 2). Each of
the five most common serotypes presented in Fig. 2 made
up 5% or more of the total amount of isolates in the 10-year
study period, and was among the top five most common
types at least five times in the 10 years of the study. Overall,
the majority of isolates were concentrated within relatively
few serotypes; depending on the year, the most common
serotypes shown in Fig. 2 accounted for 55–85% of the
isolates. The proportion of T non-typable (NT) isolates
varied between 2 and 12% annually.
The serotype distribution of Finnish invasive isolates
showed a fluctuating pattern throughout the study period.
The fluctuation was evident especially in the proportions of
the most common serotypes, T28, T1 and TB3264, and
showed that a competition existed between serotypes. The
most common serotype was T28; the proportion of isolates
reacting with anti-T28 typing serum varied annually
between 14 and 48% of the total number during the
a statistically significant increase from 1999 to 2000,
P=0?027). Comparing these results to those of a study of
Finnish isolates from 1988 to 1995, we can locate an earlier
peak of T28 between 1992 and 1996 (Muotiala et al., 1997).
In contrast, the prevalence of serotype T1 has a temporally
different cycle, and the proportion of T1 isolates in Finland
has been low since its latest peak in 1997, differing
considerably from the trend in many other countries, in
which T1/M1 isolates have recently been prevalent con-
tinuously (Muotiala et al., 1997; Hoe et al., 1999; O’Brien
et al., 2002; Li et al., 2003; Ekelund et al., 2005). Serotype
TB3264 was most prevalent in 1999 at a season between the
peaks of T28 and T1.
The proportion of serotypes grouped as ‘other type’ varied
between 11 and 35% annually (see Fig. 2 caption for the
Fig. 1. The number of notifications to the
NIDR of Finland and the total number of
referred isolates (left-hand axis). The inci-
dence of GAS bacteraemia in 1995–2004,
Finland, is given on the right-hand axis.
White bars, notifications to NIDR; black
bars, referred isolates; N, incidence.
Fig. 2. The most common T serotypes of
Finnish GAS bacteraemia isolates during
1995–2004. *Other types (in order of fre-
quency): T3/B3264, 11, 4, 13/28, 8/25, 13,
2, 9, 13/B3264, 2/28, 3, 25, 28/B3264,
Imp19, 11/8, 3/9, 5, 5/27, 5/27/44, 6, 8/
B3264, 3/13/9, 3/B3264/8, 3/B3264/28/8,
4/6, 4/28, 5/12, 5/25, 8/B3264, 8/Imp19
and 14. DNT, non-typable: no positive agglu-
tination reaction with the available T typing
3/8, 3/13, 3/13/
emm typing of invasive GAS in Finland
serotypes in this category varied annually, from five (in
1995 and 2002) to 19 (in 2004). The large increase in this
category from 2003 to 2004 was firstly due to the increase of
serotype T3 and its combinations with serotypes B3264, 13
and 9, and secondly a simultaneous decrease in serotype
TB3264 (single serum reaction), showing a shift towards
combination serotypes. It should be noted here that the T
serotyping method is associated with a certain amount of
inaccuracy and that some of the reactions with different T
antisera may be non-specific, resulting in unusual T-
agglutination patterns. When looking at the combinations
and interpreting the results, one should be aware that
different combinations of common patterns may in fact
represent the same type (Johnson et al., 2006). In reality, the
category ‘other types’ofourstudymay not be asdiverse asit
emm typing of T28 isolates
led us to investigate the clonality of these strains in greater
detail. A total of 336 isolates were emm typed; these were
isolates reacting with the T28 antiserum alone (305 isolates)
and with T28 in combination with other T typing sera (31
isolates). The combination serotypes were T13/28 (15
isolates), T2/28 (eight isolates), T28/B3264 (four isolates),
T28/11 (two isolates), T4/28 (one isolate) and T28/B3264/
28/8 (one isolate). The isolates were distributed among six
distinct emm types: emm28, emm77, emm53 (including
subtypes 53.2 and 53.4), emm87, emm2 and emm4 (Fig. 3).
The correlation of these emm types with serotype T28 has
been reported elsewhere, although emm53 has historically
been associated more with serotype T3/13/B3264 (Strakova
et al., 2005; Johnson et al., 2006; ftp://ftp.cdc.gov/pub/
The proportion of emm28 isolates was highest in 1995–97
(86–44% of all T28), declined in 1998–2000 (26–30%) and
then showed a considerable increase again in 2001–2004
(80–96%), the increase from 2000 to 2001 being statistically
significant (P<0?0005). During 1999–2000, the most
prominent type was emm77 (43–37% of all T28 isolates).
emm53 (including both subtypes), which was fairly
uncommon during the first two study years, increased to
34% of all T28 in 1998, before again becoming rare by 2001
(with a statistically significant decrease from 2000 to 2001,
P=0?023). To summarize, the emm type distribution varied
in a cyclical pattern, in which, during periods of low emm28
prevalence, emm types 77 and 53 seemed to emerge and
partially replace the emm28 type. In contrast, when emm28
became predominant again, it replaced the other emm types
almost completely. The proportion of emm87, emm4 and
emm2 remained low throughout the study period.
The M-, T- and emm-type distributions among GAS isolates
have been found to vary with time, geographic region and
disease spectrum (Johnson et al., 1992; Kaplan et al., 2001;
O’Brien et al., 2002; Ikebe et al., 2003; Shulman et al., 2004).
Our findings are in agreement with those of a Swedish
study of invasive and non-invasive isolates, in which an
increased spread of T28 with a high proportion of emm28
genotype was observed earlier in 1996–1997 (Eriksson et al.,
2003). Looking at the emm28 group alone, a very different
temporal distribution was seen in a study of Canadian
isolates,in whichthe numberof emm28isolates firststeadily
increased and then decreased over a long period from
a study of iGAS isolates from the Czech Republic, a peak in
the incidence of emm53.2 and emm53.6 strains was
observed; however, this occurred in 2001–2004, thus
differing temporally from our observations (Strakova et al.,
Within the group of isolates reacting with the single serum
T28, emm28 was the dominant type, but all the other
aforementioned emm types were found as well. It is evident
mostly emm77 or emm2. Looking at our emm28 isolates,
Fig. 3. emm types of serotype T28 GAS
Finland. The numbers represent the annual
amounts of serotype T28 isolates. The emm
typed isolates included strains reacting with
the T28 antiserum alone (n=305) and T28
in combination with other T typing sera
emm4; Dincludes emm53.2 and emm53.4.
1704Journal of Medical Microbiology 55
T. Siljander and others
almostall (99%) of them were of serotype T28 and only two
isolates were of a combination serotype T28/11. The use of
further discriminatory typing methods, such as PFGE and
MLST, as well as the determination of virulence factors
and antimicrobial susceptibility, is warranted to answer the
questionofwhether theFinnish emm28isolates inthis study
are genetically similar or whether there are different clones
withinthisgroup. InaSwedish studyofT28emm28isolates,
all isolates had the same MLST sequence type and had PFGE
patterns that differed by only one to four bands, showing
some genetic diversification (Eriksson et al., 2003). On the
other hand, M28 strains have been found to be highly
diverse in a study of prophage-associated virulence genes
(Green et al., 2005a). Furthermore, without extensive
information about the emm types within all serotypes, we
cannot exclude the possibility that we might have missed
some of the emm28 isolates when concentrating on serotype
T28 alone. emm type 28 has been observed to correlate with
T-agglutination patterns 28, 4/28, NT, 11/28, 12/28, 8/28, 3/
13/B3264 and 4 (Johnson et al., 2006). Therefore, especially
additional emm28 isolates. Should the emm28 type remain
prevalent in Finland, it will be essential to obtain more
specific information about the clonality of the isolates in
order to trace the epidemiology of these strains.
The annual incidence of iGAS bacteraemia in Finland has
increased during the last decade and follows the general
European trend. A fluctuating pattern in the serotype
distribution was evident, especially with the most common
types, T28, TB3264 and T1, showing a competition between
serotypes. emm typing of the serotype T28 isolates revealed
that several genetically distinct clones exist in this group
and that the prevalence of type emm28 in this group varied
in a cyclical pattern over time. This finding highlights
the importance of using emm typing alongside T
serotyping in the epidemiologic surveillance of iGAS
disease. Characterization of the circulating clones and
their change over time remains an essential task. To obtain a
more comprehensive view of the epidemiology of iGAS
disease and the clonality of isolates, additional typing
methods could be used to verify and complement these
We thank Aila Soininen (KTL), Saija Perovuo (KTL) and Mary Liu
(Rocky Mountain Laboratories) forexcellenttechnical assistance, Anni
Virolainen-Julkunen (KTL) and Saara Salmenlinna (KTL and
University of Helsinki) for advice, and Theresa Lamagni (Health
Protection Agency, UK) for reviewing the manuscript. This work was
supported by grants from the Academy of Finland/MICMAN Research
programme and the European Commission Framework Five pro-
gramme/Strep-EURO (QLK2-CT-2002-01398). A part of these results
have been presented in a poster at the 15th European Congress of
Clinical Microbiology and Infectious Diseases (ECCMID) 2005,
Beall, B., Facklam, R. & Thompson, T. (1996). Sequencing emm-
specific PCR products for routine and accurate typing of group A
streptococci. J Clin Microbiol 34, 953–958.
Beall, B., Gherardi, G., Lovgren, M., Facklam, R. R., Forwick, B. A. &
Tyrrell, G. J. (2000). emm and sof gene sequence variation in relation
to serological typing of opacity-factor-positive group A streptococci.
Microbiology 146, 1195–1209.
Bisno, A. L., Collins, C. M. & Turner, J. C. (1996). M proteins of
group C streptococci isolated from patients with acute pharyngitis.
J Clin Microbiol 34, 2511–2515.
Carapetis, J. R., Steer, A. C., Mulholland, E. K. & Weber, M. (2005).
The global burden of group A streptococcal diseases. Lancet Infect
Dis 5, 685–694.
Collins, C. M., Kimura, A. & Bisno, A. L. (1992). Group G
streptococcal M protein exhibits structural features analogous to
those of class I M protein of group A streptococci. Infect Immun 60,
Colman, G., Tanna, A., Efstratiou, A. & Gaworzewska, E. T. (1993).
The serotypes of Streptococcus pyogenes present in Britain during
1980–1990 and their association with disease. J Med Microbiol 39,
Doktor, S. Z., Beyer, J. M., Flamm, R. K. & Shortridge, V. D. (2005).
Comparison of emm typing and ribotyping with three restriction
enzymes to characterize clinical isolates of Streptococcus pyogenes.
J Clin Microbiol 43, 150–155.
Efstratiou, A. (2000). Group A streptococci in the 1990s. J Antimicrob
Chemother 45, 3–12.
Ekelund, K., Skinhoj, P., Madsen, J. & Konradsen, H. B. (2005).
Reemergence of emm1 and a changed superantigen profile for group
A streptococci causing invasive infections: results from a nationwide
study. J Clin Microbiol 43, 1789–1796.
Multilocus sequence typing of Streptococcus pyogenes and the relation-
ships between emm type and clone. Infect Immun 69, 2416–2427.
Eriksson, B. K., Norgren, M., McGregor, K., Spratt, B. G. &
Henriques Normark, B. (2003). Group A streptococcal infections
in Sweden: a comparative study of invasive and noninvasive
infections and analysis of dominant T28 emm28 isolates. Clin
Infect Dis 37, 1189–1193.
Facklam, R., Beall, B., Efstratiou, A. & 13 other authors (1999).
emm typing and validation of provisional M types for group A
streptococci. Emerg Infect Dis 5, 247–253.
Facklam, R. F., Martin, D. R., Lovgren, M. & 8 other authors (2002).
Extension of the Lancefield classification for group A streptococci by
addition of 22 new M protein gene sequence types from clinical
isolates: emm103 to emm124. Clin Infect Dis 34, 28–38.
Fischetti, V. A. (1989). Streptococcal M protein: molecular design
and biological behavior. Clin Microbiol Rev 2, 285–314.
Green, N. M., Beres, S. B., Graviss, E. A., Allison, J. E., McGeer, A. J.,
Vuopio-Varkila, J., LeFebvre, R. B. & Musser, J. M. (2005a). Genetic
diversity among type emm28 group A Streptococcus strains causing
invasive infections and pharyngitis. J Clin Microbiol 43, 4083–4091.
Green, N. M., Zhang, S., Porcella, S. F., Nagiec, M. J., Barbian, K. D.,
Beres, S. B., LeFebvre, R. B. & Musser, J. M. (2005b). Genome
sequence of a serotype M28 strain of group A Streptococcus: potential
new insights into puerperal sepsis and bacterial disease specificity.
J Infect Dis 192, 760–770.
Hoe, N. P., Nakashima, K., Lukomski, S. & 15 other authors (1999).
Rapid selection of complement-inhibiting protein variants in group
A Streptococcus epidemic waves. Nat Med 5, 924–929.
emm typing of invasive GAS in Finland
Ikebe, T., Murai, N., Endo, M. & 14 other authors (2003). Changing Download full-text
prevalent T serotypes and emm genotypes of Streptococcus pyogenes
isolates from streptococcal toxic shock-like syndrome (TSLS)
patients in Japan. Epidemiol Infect 130, 569–572.
Johnson, D. R. & Kaplan, E. L. (1993). A review of the correlation of
T-agglutination patterns and M-protein typing and opacity factor
production in the identification of group A streptococci. J Med
Microbiol 38, 311–315.
Johnson, D. R., Stevens, D. L. & Kaplan, E. L. (1992). Epidemiologic
analysis of group A streptococcal serotypes associated with severe
systemic infections, rheumatic fever, or uncomplicated pharyngitis.
J Infect Dis 166, 374–382.
Johnson, D. R., Kaplan, E. L., Sramek, J., Bicova, R., Havlicek, J. &
Havlickova, H. (1996). Laboratory diagnosis of group A streptococcal
infections. Geneva, World Health Organization.
Johnson, D. R., Kaplan, E. L., VanGheem, A., Facklam, R. R. & Beall, B.
(2006). Characterization of group A streptococci (Streptococcus
pyogenes): correlation of M-protein and emm-gene type with T-
protein agglutination pattern and serum opacity factor. J Med
Microbiol 55, 157–164.
Kaplan, E. L., Wotton, J. T. & Johnson, D. R. (2001). Dynamic
epidemiology of group A streptococcal serotypes associated with
pharyngitis. Lancet 358, 1334–1337.
Lamagni, T., Efstratiou, A., Vuopio-Varkila, J., Jasir, A. & Schalen, C.
(2005). The epidemiology of severe Streptococcus pyogenes associated
disease in Europe. Euro Surveill 10, 179–184.
Lancefield, R. C. (1962). Current knowledge of type-specific M
antigens of group A streptococci. J Immunol 89, 307–313.
Li, Z., Sakota, V., Jackson, D., Franklin, A. R. & Beall, B. (2003).
Array of M protein gene subtypes in 1064 recent invasive group A
streptococcus isolates recovered from the active bacterial core
surveillance. J Infect Dis 188, 1587–1592.
Maxted, W. R., Widdowson, J. P. M., Fraser, C. A. M., Ball, L. C. &
Bassett, D. C. J. (1973). The use of the serum opacity reaction in the
typing of group A streptococci. J Med Microbiol 6, 83–90.
Bessen, D. E. (2004). Multilocus sequence typing of Streptococcus
pyogenes representing most known emm types and distinctions among
subpopulation genetic structures. J Bacteriol 186, 4285–4294.
Moody, M. D., Padula, J., Lizana, D. & Hall, C. T. (1965).
Epidemiologic characterization of group A streptococci by T-
agglutination and M-precipitation tests in the public health
laboratory. Health Lab Sci 2, 149–162.
Shetzigovsky, I., Ravins, M., Korenman, Z., Cohen-Poradosu, R.
& Nir-Paz, R. (2003). emm typing of M nontypeable invasive group A
streptococcal isolates in Israel. J Clin Microbiol 41, 4655–4659.
A.E., Hidalgo-Grass, C.,Dan-Goor,M., Jaffe,J.,
Muotiala, A., Seppala, H., Huovinen, P. & Vuopio-Varkila, J. (1997).
Molecular comparison of group A streptococci of T1M1 serotype
from invasive and noninvasive infections in Finland. J Infect Dis 175,
O’Brien, K. L., Beall, B., Barrett, N. L. & 8 other authors (2002).
Epidemiology of invasive group A Streptococcus disease in the United
States, 1995–1999. Clin Infect Dis 35, 268–276.
Podbielski, A., Melzer, B. & Lu ¨tticken, R. (1991). Application of the
polymerase chain reaction to study the M protein(-like) gene family
in beta-hemolytic streptococci. Med Microbiol Immunol 180,
Raymond, J., Schlegel, L., Garnier, F. & Bouvet, A. (2005). Molecular
characterization of Streptococcus pyogenes isolates to investigate an
outbreak of puerperal sepsis. Infect Control Hosp Epidemiol 26,
Saunders, N. A., Hallas, G., Gaworzewska, E. T., Metherell, L.,
Efstratiou, A., Hookey, J. V. & George, R. C. (1997). PCR-enzyme-
linked immunosorbent assay and sequencing as an alternative to
serology for M-antigen typing of Streptococcus pyogenes. J Clin
Microbiol 35, 2689–2691.
Rummukainen, M., Holm, S. E. & Huovinen, P. (1994). Evaluation
of methods for epidemiologic typing of group A streptococci. J Infect
Dis 169, 519–525.
H., Vuopio-Varkila,J., Osterblad, M.,Jahkola,M.,
Shulman, S. T., Tanz, R. R., Kabat, W. & 7 other authors (2004).
Group A streptococcal pharyngitis serotype surveillance in North
America, 2000–2002. Clin Infect Dis 39, 325–332.
Single, L. A. & Martin, D. R. (1992). Clonal differences within M-
types of the group A Streptococcus revealed by pulsed field gel
electrophoresis. FEMS Microbiol Lett 70, 85–89.
Strakova, L., Motlova, J., Jakubu, V., Urbaskova, P. & Kriz, P. (2005).
Molecular analysis of selected Czech Group A Streptococcus strains.
Poster at the XVIth Lancefield International Symposium on
Streptococci and Streptococcal Diseases, Cairns, Australia, 25–29
Tyrrell, G. J., Lovgren, M., Forwick, B., Hoe, N. P., Musser, J. M. &
Talbot, J. A. (2002). M types of group A streptococcal isolates
submitted to the National Centre for Streptococcus (Canada) from
1993 to 1999. J Clin Microbiol 40, 4466–4471.
1706Journal of Medical Microbiology 55
T. Siljander and others