Abstract
Group A streptococcus (GAS) is an important cause of morbidity and mortality worldwide. Surveillance of emm types has important implications, as it can provide baseline information for possible implementation of vaccination. A total of 1,349 GAS pediatric isolates were collected during a 7-year period (2007 to 2013); emm typing was completed for 1,282 pharyngeal (84%) or nonpharyngeal (16%) isolates, and emm clusters and temporal changes were analyzed. Thirty-five different emm types, including 14 subtypes, were identified. The most prevalent emm types identified were 1 (16.7%), 12 (13.6%), 77 (10.9%), 4 (10.8%), 28 (10.4%), 6 (6.8%), 3 (6.6%), and 89 (6.6%), accounting for 82.3% of total isolates. Rheumatogenic emm types comprised 16.3% of total isolates. The emm types 12, 4, and 77 were more prevalent among pharyngeal isolates, and the emm types 1, 89, 6, 75, and 11 were more prevalent among nonpharyngeal isolates. The emm types identified belonged to 13 emm clusters, and the 8 most prevalent clusters comprised 97% of all isolates. There were statistically significant decreases in the prevalence of emm types 12, 4, 5, and 61 and increases in the prevalence of emm types 89, 75, and 11, compared with the period 2001 to 2006. The proposed 30-valent GAS vaccine, which is currently in preclinical studies, encompasses 97.2% of the emm types detected in our study and 97.4% of the erythromycin-resistant strains. In addition, it includes 93.3% of the emm types involved in bacteremia. A much greater diversity of GAS emm types was identified in our area than described previously. Seasonal fluctuations and the introduction of new emm types were observed. Continuous surveillance of emm types is needed in order to evaluate the possible benefits of an M protein-based GAS vaccine.
INTRODUCTION
Streptococcus pyogenes (group A streptococcus [GAS]) is an important cause of morbidity and mortality worldwide (1). GAS causes a broad spectrum of diseases, ranging from mild to life-threatening invasive infections. GAS infections can also lead to immune-mediated sequelae such as rheumatic fever and glomerulonephritis, with long-term complications (2, 3).
In order to yield such a diversity of clinical manifestations, GAS possesses a large number of virulence mechanisms, with a high degree of variability in virulence determinants among different GAS serotypes (4, 5). The cell wall-associated M protein, encoded by the emm gene, is a major antigenic epitope and virulence factor of GAS and forms the basis for the serotyping of GAS isolates (6). The M protein acts as an epithelial adhesion factor, inhibits phagocytosis, and allows the organism to overcome innate immune responses (7, 8). The N terminus consists of a highly variable amino acid sequence, resulting in antigenic diversity, and is the basis for a nucleotide-based emm-typing scheme (9). There are at least 220 different emm types and subtypes of GAS, and new types are still being identified (8, 10, 11). Temporal, geographic, and seasonal variations in the dominant strains are well described and can result in variable epidemiological characteristics of disease (12). Recently, an emm cluster system based on strong phylogenetic support has been described; it serves as a functional classification scheme for GAS M proteins and can support vaccine design and evaluation (9, 11).
Vaccine prevention of GAS infections and their immunological complications has been a goal of researchers for decades, because GAS infections consume substantial economic and health resources (1, 2). Vaccine development has met several obstacles, including the theoretical risk that vaccines may induce acute rheumatic fever or glomerulonephritis through molecular mimicry or other autoimmune mechanisms (2). Several candidates for vaccines against GAS infection are in various stages of preclinical or clinical development (2, 13). These vaccines are based on several type-specific M proteins, on conserved M protein epitopes, or on other GAS antigens, such as fusions of streptococcal pyrogenic exotoxins (2, 14, 15). The aim of the present study was to investigate differences in emm type prevalence and diversity, through time and in different clinical syndromes, that could possibly affect the benefits of an M protein-based GAS vaccine.
MATERIALS AND METHODS
The study was conducted prospectively at Aghia Sophia Children's Hospital (Athens, Greece), which is the largest tertiary pediatric hospital in Greece and serves approximately 40% of children residing in the Athens metropolitan area, as well as patients referred from other parts of Greece. GAS isolates from nonpharyngeal infections were recovered from children who were hospitalized during a 7-year period (January 2007 to December 2013). Isolates from pharyngitis cases were collected twice a month from infectious disease fellows in the emergency department, during the same period. The study was approved by the institutional review board of Aghia Sophia Children's Hospital.
The identification of the isolates as GAS was confirmed by standard techniques, including colony morphology, typical beta-hemolysis on 5% sheep blood agar (Becton Dickinson, Franklin Lakes, NJ), inhibition screening tests using 0.04-U bacitracin disks (Becton Dickinson), and positive pyrrolidonyl aminopeptidase test results (LifeSign, Somerset, NJ). The presence of Lancefield A antigen was confirmed by using a commercially available agglutination technique (Slidex Streptokit; bioMérieux, Marcy l'Etoile, France). The GAS isolates were stored at −70°C. Disk diffusion methods were used to determine the susceptibilities to commonly used antibiotics, in accordance with Clinical and Laboratory Standards Institute guidelines (16).
Streptococcal DNA was extracted according to CDC laboratory protocols for S. pyogenes (http://www.cdc.gov/streplab/protocols.html). The emm types were determined by amplification of the M protein gene (emm) and sequencing using an automated sequencer (Avant 3100; Applied Biosystems, Foster City, CA). All protocols and assignment of emm types and subtypes were as described for the S. pyogenes emm sequence database (http://www.cdc.gov/streplab/protocol-emm-type.html) (17). The assignment of emm clusters was based on the CDC database (http://www.cdc.gov/streplab/downloads/distribution-emm-types.pdf) (9, 18). Comparison with the distribution of emm types circulating in 2003 to 2006 was performed using previously published data from our laboratory (19).
Data were analyzed by using SPSS v.19.0 (IBM Inc.) and XLSTAT v.5.03 (Addinsoft). Differences in the distributions of emm types were analyzed by the chi-square test or by Fisher's exact test, when appropriate; emm trends over time were analyzed by the Cochran-Armitage trend test. All P values reported are two-tailed, and statistical significance was set at 0.05. Diversity was calculated through Simpson's index by using an on-line tool (http://darwin.phyloviz.net/ComparingPartitions). The value of this index ranges from 0 to 1, and greater values indicate greater diversity.
RESULTS
Sample and patient characteristics.
A total of 1,324 strains were collected during the 7-year period, including 1,118 (84.4%) from the throat, 98 (7.3%) from ear fluid, 33 (2.4%) from blood, 22 (1.7%) from tonsillar abscesses, 21 (1.6%) from skin infections, 14 (1%) from osteomyelitis or synovial fluid, 12 from vaginal fluid, and 6 from pleuritic fluid. The median age of the children was 65 months (interquartile range [IQR], 47 to 96 months), and 51.8% were boys; emm typing was completed for 1,282 isolates, including 1,080 pharyngeal (84.2%) and 202 nonpharyngeal (15.8%) isolates.
emm types.
The emm type distribution for 1,282 GAS strains is shown in Fig. 1. Thirty-five different emm types, including 15 subtypes, were identified. The most prevalent emm types identified were 1 (16.7%), 12 (13.6%), 77 (10.9%), 4 (10.8%), 28 (10.4%), 6 (6.8%), 3 (6.6%), and 89 (6.6%), accounting for 82.3% of the total population. The following emm subtypes were detected: 1.14 (n = 1), 2.1 (n = 9), 2.2 (n = 2), 4.1 (n = 2), 5.5 (n = 4), 5.8193 (n = 3), 6.63 (n = 64), 12.17 (n = 1), 18.1 (n = 2), 18.7 (n = 6), 22.1 (n = 1), 22.3 (n = 2), 78.3 (n = 2), 80.1 (n = 1), and 82.1 (n = 2).
FIG 1.
Distribution of emm types among 1,282 Streptococcus pyogenes isolates during a 7-year period (2007 to 2013). The emm types on the right are not included in the 30-valent vaccine currently in preclinical studies.
The proposed 30-valent GAS vaccine (currently in preclinical trials) (13, 20) encompasses 97.27% of emm types identified in our region and 97.4% of erythromycin-resistant strains. In addition, it includes 93.3% of emm types involved in bacteremia. We did not detect 7 emm types that are included in the proposed vaccine, namely, emm14.3, emm19, emm24, emm49, emm83.1, emm92, and emm114. We identified 13 emm types that are not included in the vaccine and account for 2.73% of all strains and 2.6% of erythromycin-resistant strains (Fig. 1).
Annual circulation of emm types.
Significant differences in the annual circulation of specific GAS emm types were detected during the study years (Fig. 2). The incidence of emm89 increased from 1.3% in 2007 to 16.2% in 2013 (P < 0.0001 for trend, Cochran-Armitage trend test). The opposite trend was detected for emm77 (P < 0.001), emm4 (P = 0.006), and emm6 (P = 0.03) (P values for trend, Cochran-Armitage trend test). The prevalence of specific emm types, i.e., emm3 (P = 0.7), emm28 (P = 0.53), emm12 (P = 0.06), emm1 (P = 0.7), and the rest of the emm types (P = 0.6), was relatively stable. Analysis of Simpson's index of diversity year by year found the lowest value in 2007 (0.886 [95% confidence interval [CI], 0.875 to 0.897]) and the highest values in 2008 (0.914 [95% CI, 0.894 to 0.933]) and in 2013 (0.914 [95% CI, 0.901 to 0.927]).
FIG 2.
Annual prevalence of Streptococcus pyogenes emm types with statistically significant differences in distribution during 2007 to 2013. Increased prevalence was detected for emm89 (P < 0.0001) and decreased prevalence for emm77 (P < 0.001), emm4 (P = 0.006), and emm6 (P = 0.03) (Cochran-Armitage trend test).
Rheumatogenic types.
The classic rheumatogenic GAS types (21), including emm3 (6.6%), emm5 (2%), emm6 (6.8%), emm18 (0.7%), and emm29 (0.23%), represented 16.3% of all isolates; no emm14 or emm19 was identified in this data set. A total of 93.5% of those isolates were macrolide sensitive. Rheumatogenic GAS types represented 15% of pharyngeal isolates and 21% of nonpharyngeal isolates (P = 0.04).
emm clusters.
Analysis of emm types according to emm cluster classification indicated that 35 emm types belonged to 13 emm clusters (Table 1). The most prevalent emm clusters were E4 (33.3%), A-C3 (16.6%), A-C4 (13.6%), E1 (11%), M6 (6.8%), A-C5 (6.6%), E6 (5.5%), and E3 (3.5%), together accounting for 97% of all isolates. Eight emm clusters (A-C3, A-C4, M6, A-C5, M5, M18, M29, and M74) comprised only one emm type.
TABLE 1.
Distribution of GAS emm types according to emm cluster for years 2007 to 2013
| emm clustera | emm type(s) | No. (%) of isolates |
|---|---|---|
| E4 | 77, 28, 22, 89, 2, 8, 73, 40, 84 | 427 (33.3) |
| A-C3 | 1 | 213 (16.6) |
| A-C4 | 12 | 174 (13.6) |
| E1 | 4, 78 | 141 (11.0) |
| M6 | 6 | 87 (6.8) |
| A-C5 | 3 | 85 (6.6) |
| E6 | 75, 11, 81, 65, 48 | 68 (5.5) |
| E3 | 87, 44, 118, 58, 82, 9, 15, 25, 61 | 45 (3.5) |
| M5 | 5 | 26 (2.0) |
| M18 | 18 | 9 (0.7) |
| M29 | 29 | 3 (0.2) |
| D4 | 80, 108 | 3 (0.2) |
| M74 | 74 | 1 (0.1) |
Thirteen emm clusters were identified among 1,282 isolates.
emm types among nonpharyngeal isolates.
Comparing the distribution of GAS emm types in pharyngeal and nonpharyngeal isolates, we found that emm types 12, 4, and 77 were more prevalent among pharyngeal isolates and emm types 1, 89, 6, 75, and 11 were more prevalent among nonpharyngeal isolates (Fig. 3). The most prevalent emm types from different sources are presented in Table 2. The most common emm types in GAS isolates from the throat were emm12 (15.7%) and emm1 (15.6%), from bacteremia emm1 (43.3%), emm77 (10%), and emm6 (10%), and from ear fluid emm1 (16.5%) and emm89 (12.4%). Comparing the distributions of emm types according to the source of isolation, we found statistically significant differences between pharyngeal isolates and isolates from bacteremia, ear fluid, skin infections, arthritis/osteomyelitis, or peritonsillar abscesses (P < 0.001).
FIG 3.
emm type distribution among pharyngeal (n = 1,080) and nonpharyngeal (n = 202) isolates. ★, statistically significant differences in distribution (chi-square test, P < 0.05).
TABLE 2.
Prevalent emm types in Streptococcus pyogenes isolates from different clinical syndromes
| Type of infection | No. of isolates (% of total) | Prevalent emm type(s) (% of type of infection) | Pa |
|---|---|---|---|
| Pharyngitis | 1,080 (84.2) | 12 (15.7), 1 (15.6), 4 (11.8), 77 (11.7), 28 (10.7), 3 (6.8) | |
| Ear infection | 98 (7.6) | 1 (16.5), 89 (12.4), 28 (11.3), 6 (8.3), 75 (7.2) | <0.001 |
| Bacteremia | 30 (2.4) | 1 (43.3), 6 (10), 77 (10), 12 (6.7), 3 (6.7) | <0.001 |
| Tonsillar abscess | 22 (1.7) | 3 (18.2), 89 (13.6), 1 (9.1), 2 (9.1), 28 (9.1), 4 (9.1) | 0.005 |
| Skin infection | 20 (1.5) | 28 (14.3), 89 (14.3), 1 (9.5), 75 (9.5) | <0.001 |
| Arthritis/osteomyelitis | 14 (1) | 1 (30.8), 11 (23.1), 2 (7.7), 28 (7.7), 3 (7.7) | <0.001 |
| Vaginitis | 12 (0.9) | 89 (36.4), 1 (27.3), 6 (18.2) | 0.32 |
| Pleuritic fluid | 6 (0.5) | 1 (100) | 0.84 |
P values indicate differences in distribution, compared with pharyngeal isolates (chi-square test).
Comparison of emm types from 2003 to 2006 and 2007 to 2013.
The distributions of emm types in two different time periods are presented in Table 3. During the latter period, there were statistically significant decreases in the prevalence of emm types 12, 4, 5, and 61 and increases in the prevalence of emm 89, 75, and 11. The Simson's index of diversity found for the years 2007 to 2013 was 0.907 (95% CI, 0.901 to 0.913) and that for the years 2003 to 2006 was 0.876 (95% CI, 0.869 to 0.883), indicating greater diversity of emm types in the recent study period.
TABLE 3.
Comparison of distributions of prevalent emm types in two time periods (2001 to 2006 and 2007 to 2013) at our center
| emm type | % of total |
Pb | |
|---|---|---|---|
| 2003–2006 (n = 1,160)a | 2007–2013 (n = 1,282) | ||
| 1 | 17.3 | 16.7 | 0.7 |
| 12 | 17.8 | 13.6 | 0.006 |
| 4 | 14.2 | 10.9 | 0.01 |
| 77 | 13.6 | 10.9 | 0.05 |
| 28 | 12.7 | 10.4 | 0.09 |
| 3 | 5.5 | 6.7 | 0.24 |
| 89 | 0 | 6.6 | <0.001 |
| 6 | 5.5 | 6.8 | 0.20 |
| 2 | 3.1 | 2.9 | 0.78 |
| 75 | 0 | 2.7 | <0.001 |
| 11 | 0.1 | 2.1 | <0.001 |
| 5 | 3.4 | 2 | 0.03 |
| 22 | 2.2 | 1.7 | 0.39 |
| 87 | 0.3 | 0.9 | 0.08 |
| 61 | 3.8 | 0.9 | <0.001 |
| 44 | 0 | 0.8 | 0.005 |
The distribution of emm types for 2003 to 2006 is derived from the report by Michos et al. (19).
P values were calculated with the chi-square test or Fisher's exact test.
DISCUSSION
Analysis of the dynamics of the circulation of S. pyogenes isolates is complex and can be affected by multiple factors such as climate, antibiotic pressure, population immunity, and strain characteristics (1). This is the first report from our area regarding characterization of GAS emm types and emm clusters from pharyngeal and nonpharyngeal GAS isolates for the time period after 2006.
Although more than 220 emm types have been described to date, only a few tend to predominate within a community. In our study, we identified 35 different emm types and, among them, 15 emm subtypes that are much more diverse than the 18 emm types identified in our area in 2001 to 2006 (19). In a study from western Greece regarding pediatric pharyngeal isolates for the period from 1999 to 2005, 28 different emm types were recognized (22). In another study regarding invasive isolates from children and adults for the years 2003 to 2007, 31 different emm types were recognized, with predominance of emm1 (28.2%) and emm12 (8.5%) (23).
Although it appears from the present study that there is more diversity in emm types than previously reported, our findings are much less diverse than data from other countries, such as the United States (56 emm types), Bali (70 emm types), Fiji (52 emm types), and Ethiopia (76 emm types) (24–27). In a recent study from Spain, 29 emm types and 45 emm subtypes were recognized (28).
In a systematic review of global emm type distribution, it was found that there is greater diversity of emm types in Africa and the Pacific region than in other regions, with a lack of dominant types (12). This may be explained because different clinical presentations of GAS infections in these regions, especially endemic impetigo, contribute different emm type profiles. The variety of emm types on different continents makes the implementation of a universal M protein-based vaccine difficult. Increased immigration and populations moving from these regions to European countries could possibly affect the circulating emm types and the dynamics of S. pyogenes-associated diseases. Changes in the predominant circulating emm types within relatively short time periods were shown to occur in individual regions in the United States, although this was explained by the replacement of common emm types with other common types from other regions in the United States (26).
There were differences in the annual circulation of different emm types during the study period, as some emm types (emm1, emm4, and emm77) decreased and new emm types appeared. Compared with the years 2001 to 2006, 18 new emm types were identified; in particular, emm89, which was absent during the previous period, gradually increased significantly. In Spain, emm89 behaved like an epidemic emm type, and its relative frequency increased in the last 3 years, affecting especially older patients (28). Recently, an outbreak of a single clone of emm89 was described in China (29).
Surveillance for severe cases of S. pyogenes infections diagnosed during 2003 and 2004 was undertaken in 11 countries across Europe through the Strep-EURO program (30). A wide diversity of emm types (n = 104) was found among 4,353 S. pyogenes clinical isolates, but the emm type distributions varied widely among participating countries. The most prevalent types were emm1 (19%), emm28 (12%), emm3 (10%), and emm89 (8%), which were also found in high prevalence in our study (30). Since the middle 1980s, there have been increasing numbers of reports describing severe manifestations of GAS infections; however, the factors underlying the worldwide resurgence of this pathogen remain unknown (3). Rates of reported severe GAS infections during the Strep-EURO project varied, reaching 3 cases/100,000 population in northern European countries, with lower incidences in southern European countries (31). In a study from Greece, Zachariadou et al., comparing invasive GAS isolates from children and adults, found that the main predisposing factors for severe disease in children were varicella (25%) and streptococcal pharyngotonsillitis (19.8%). One-half of the severe GAS cases involved previously healthy children, and 13/96 children (13.6%) required intensive care unit (ICU) admission (23).
A new emm cluster typing system has been recently proposed for group A streptococcus (GAS) (9). This system classifies most of the 223 emm types into 48 functional emm clusters, containing closely related M proteins that share structural properties (9, 11). The advantage of emm clusters is that they help to predict the virulence potential of any GAS isolate by ascribing M protein binding attributes to emm types belonging to the same emm cluster (8, 9). This system correlates with M protein vaccine antigen contents and serves as a framework for investigations of immunological cross-protection between emm types (9, 32, 33). Therefore, the emm cluster system provides a working hypothesis for the recently discovered but unexplained cross-protection between different emm types (13, 20).
In our data set, we identified 13 emm clusters; among them, 8 clusters comprised 97% of the isolates. Applying the emm cluster system to North American pharyngitis isolates, Shulman et al. found that 56 emm types in the United States and 33 emm types in Canada were assigned to 18 and 14 emm clusters, respectively (34). The emm cluster system has been used to analyze the epidemiology of GAS in the Pacific region, which is characterized by large GAS disease burden and a great variety of circulating emm types (12, 35). The emm cluster system identified epidemiological similarities across the Pacific region and highlighted vaccine target priorities (18).
The development of a safe, effective, affordable vaccine designed to prevent GAS infections has been considered an attractive approach for a long time (2, 20). Vaccine development has faced obstacles related to the complex epidemiological features of GAS infections, especially in developing countries (12). A greater burden of GAS disease occurs in developing countries, particularly those located in the tropics, compared with industrialized nations (1). The spectrum of GAS disease also differs between developed and developing countries (12). In industrialized countries, a massive number of cases of GAS pharyngitis leads to significant economic impact, and invasive disease leads to a significant number of deaths (36). GAS vaccines also face safety concerns based on the theoretical possibility that the vaccines may elicit autoimmune responses that could mimic acute rheumatic fever or other sequelae of GAS infections (2).
Several GAS vaccines are in various phases of preclinical or clinical development. The potential efficacy of vaccines based on the M protein is limited by their suitability for local epidemiological situations, and the molecular epidemiological features of GAS are very different in tropical settings than in temperate industrialized countries (26). A new 30-valent M protein-based vaccine, which represents the evolution of a previously tested 26-valent vaccine, is currently in the preclinical stage of development (13). This vaccine contains protective M protein peptides from serotypes of GAS that account for 98% of all cases of pharyngitis in the United States and Canada, 90% of invasive disease in the United States, and 78% of invasive disease in Europe (27, 30, 37). Preclinical studies to date have shown that the vaccine evokes bactericidal antibodies against all 30 vaccine serotypes. In addition, significant levels of cross-opsonic bactericidal antibodies have been observed against 24 of 40 nonvaccine serotypes tested to date (13). These results suggest that the potential efficacy of the 30-valent vaccine may extend well beyond the constituent M peptides. This 30-valent GAS vaccine would potentially cover almost 97.2% of the emm types detected in our study and 97.4% of the erythromycin-resistant strains. This is much higher than the 80% coverage with the 26-valent vaccine estimated in previous studies (19, 22).
The present study has limitations, as it is a single-center study and the GAS isolates are representative of a specific geographic area. As this was a laboratory-based study, we could not determine the real incidence of streptococcal disease in our region. Identified differences in the distributions of emm types among clinical syndromes should be interpreted with caution, as there is great overlap of most emm types from different clinical sources and we did not investigate clonal similarities and differences among emm types.
Because S. pyogenes is an important cause of morbidity and mortality worldwide, the development of an effective safe vaccine will give new opportunities for primary prevention strategies for GAS-associated diseases. Continuous surveillance of GAS molecular epidemiological patterns in different regions and time periods could have implications for vaccine design.
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