Molecular typing of Streptococcus pyogenes from remote Aboriginal communities where rheumatic fever is common and pyoderma is the predominant streptococcal infection

Aboriginal Australians in remote communities have high rates of rheumatic heart disease (RHD); yet pharyngitis is reportedly rare whilst pyoderma is common. Some strains of group A streptococci (GAS) have preference for the throat and others for the skin depending on M protein type. A study in three remote communities provided 350 GAS isolates for emm sequence typing, 244 were also emm pattern typed. There was 100% correlation between emm sequence and pattern type. Patterns D and E (non-throat tropic) made up 71% of throat and 87% of skin isolates although patterns A-C (throat tropic) were more common in the throat than the skin (RR 2.3, 95% CI 1.4-3.8) whilst the opposite was found for pattern D (RR 2.2, 95% CI 1.7-3.0). Pattern E favoured the throat (RR 1.4, 95% CI 1.1-1.8). Where environmental factors predispose to skin infection, emm pattern types D and E prevail, whatever the recovery site.     

Group A streptococcal infections: trend and strain emm typing in an area of central Italy, 1985-2002

A retrospective study of group A streptococcal (GAS) infections was performed for the period 1985-2002 in an area of central Italy. Although very severe diseases such as streptococcal toxic shock syndrome (STSS) were observed, a general increase in invasive infections was not found. Isolates of GAS were classified by M protein genotyping (emm typing) and analysed according to their origin from invasive and non-invasive infections. The predominant emm types were types 1, 4 and 12, followed by types 3, 6 and 28. During the study period the proportion of isolates of types 1 and 12 fell, while other types (3, 6, 22, 28 and 77) appeared. Isolates from invasive and non-invasive infections shared several emm types; however, most invasive strains belonged to five types only (types 1, 4, 12, 28 and 77), while non-invasive isolates were generally more heterogeneous.     

Group A Streptococcus emm gene types in pharyngeal isolates, Ontario, Canada, 2002-2010

Group A Streptococcus (GAS) is a human-adapted pathogen that causes a variety of diseases, including pharyngitis and invasive infections. GAS strains are categorized by variation in the nucleotide sequence of the gene (emm) that encodes the M protein. To identify the emm types of GAS strains causing pharyngitis in Ontario, Canada, we sequenced the hypervariable region of the emm gene in 4,635 pharyngeal GAS isolates collected during 2002-2010. The most prevalent emm types varied little from year to year. In contrast, fine-scale geographic analysis identified inter-site variability in the most common emm types. Additionally, we observed fluctuations in yearly frequency of emm3 strains from pharyngitis patients that coincided with peaks of emm3 invasive infections. We also discovered a striking increase in frequency of emm89 strains among isolates from patients with pharyngitis and invasive disease. These findings about the epidemiology of GAS are potentially useful for vaccine research.     

Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia

A steady increase in macrolide resistance in Streptococcus pyogenes, group A streptococci (GAS) was reported in Serbia during 2004–2009 (9.9%). However, there are no data on the molecular epidemiology of pharyngeal macrolide resistance GAS (MRGAS) isolates. Therefore, the aims of this first nationwide study were to examine the prevalence of macrolide resistance in Serbian GAS and to determine their resistance phenotypes, genotypes and clonal relationships. Overall 3893 non-duplicate pharyngeal S. pyogenes isolates from outpatients with GAS infection were collected throughout country during 2008 and 2009. Among 486 macrolide resistant pharyngeal isolates collected, 103 were further characterized. Macrolide resistance phenotypes and genotypes were determined by double-disk diffusion test and PCR, respectively. Strain relatedness was determined by emm typing, multilocus sequence typing (MLST), multilocus variable tandem repeat analysis (MLVA), phage profiling (PP) and virulence factor profiling (VFP). Overall, macrolide resistance among GAS isolates in Serbia was 12.5%. M phenotype was the most common (71.8%), followed by iMLS (18.4%) and cMLS (9.7%). Three clonal complexes – emm75/mefA/ST49, emm12/mefA/ST36 and emm77/ermA/tetO/ST63 comprised over 90% of the tested strains. Although MLVA, PP and VFP distinguished 10, 20 and 12 different patterns, respectively, cluster analysis disclosed only small differences between strains which belonged to the same emm/ST type. Our data indicate dominance of three major internationally widely disseminated macrolide resistant clones and a high genetic homogeneity among the Serbian MRGAS population. Continued surveillance of macrolide resistance and clonal composition in MRGAS in Serbia in future is necessary to determine stability of MRGAS clones and to guide therapy strategies.     

The wound care team: a new source of group a streptococcal nosocomial transmission.

BACKGROUND: In August 2001, the Centers for Disease Control and Prevention (CDC) notified the Texas Department of Health (TDH) of an unusually high number of wounds infected with group A streptococci (GAS) in an acute care facility. The TDH initiated an investigation, ultimately identifying 28 cases of non-pharyngeal, non-community-acquired GAS that had occurred between December 2000 and August 2001 and resulted in 3 deaths and 4 nonfatal cases of invasive disease. Ten specimens were sent to the CDC for emm typing; all isolates were emm type 114. However, the source of the outbreak could not be confirmed through laboratory testing at that time. METHODS: A case-control study was conducted comparing the 10 case-patients with 52 control-patients with wounds that were not infected with GAS. Age, gender, type of wound, underlying medical conditions, and treatment by the wound care team were examined for association with GAS infection. RESULTS: The odds of having wound care team treatment versus not having it were 424.2 (95% confidence interval, 19.0 to 9,495.2) among case-patients when compared with control-patients. No other risk factor showed this magnitude of association. CONCLUSIONS: This study provided overwhelming epidemiologic evidence that the wound care team was the means of transmission. One year later, when two patients receiving wound care were concurrently diagnosed as having GAS, a member of the wound care team was found to be GAS positive for the matching emm type. This is the first report of a GAS hospital outbreak linked to a wound care team.     

Evaluation of synthetic,M type-specific peptides as antigens in a multivalent group A streptococcal vaccine

The recent development of emm gene sequence-based typing methodology has allowed group A streptococci (GAS) M serotype prevalence data to be determined. This information has been used to identify the components of a multivalent M protein peptide vaccine that could theoretically prevent most of the GAS-mediated diseases in the USA. In this study, we have evaluated in mice the immunogenicity and protective ability of multiple synthetic, M type-specific peptides, derived from the N-termini of three prevalent GAS serotypes (three peptides per serotype, total of nine peptides). At least one peptide, representing each of the three M types tested, was immunogenic. Five of the nine synthetic peptides tested, elicited an immune response in mice, and sera raised against four of the peptides, all possessed functional activity as demonstrated in a bactericidal assay. In vivo nasopharyngeal challenge experiments were carried out with peptides from the M1 (peptide M1-3) and M3 (peptide M3-2) proteins induced in vivo immune protection by reducing intranasal carriage.Reduction in colonization for M1-3 and M3-2 was 90% (P=0.02) and 66% (P<0.17), respectively. A reduction in colonization of 67% (P=0.03) was observed for M3-2 immunized mice when M43, a heterologous serotype, was used as the challenge strain. These results show the utility of synthetic, M type-specific peptides as antigens in a multivalent GAS vaccine.     

Epidemiology of Streptococcus dysgalactiae subsp. equisimilis in tropical communities, Northern Australia

Streptococcus dysgalactiae subsp. equisimilis (groups C and G streptococci [GCS/GGS]) is an increasingly recognized human pathogen, although it may follow indirect pathways. Prospective surveillance of selected households in 3 remote Aboriginal communities in Australia provided 337 GCS/GGS isolates that were emm sequence-typed. Lancefield group C isolates (GCS) were localized to specific households and group G isolates (GGS) were more evenly distributed. GCS/GGS was more frequently recovered from the throat than group A streptococci (GAS [S. pyogenes]) but rarely recovered from skin sores, and then only with Staphylococcus aureus or GAS. Symptomatic GGS/GGC pharyngitis was also rare. Specific emm sequence types of GCS/GGS did not appear to cycle through the communities (sequential strain replacement) in a manner suggesting acquisition of type-specific immunity. These communities already have high levels of streptococcal and poststreptococcal disease. GCS/GGS may increase in importance as it acquires key virulence factors from GAS by lateral gene transfer.     

猩红热患儿A族链球菌emm基因分型及地域与毒力基因分布相关研究

 目的 探讨猩红热儿童分离来源的A族链球菌（GAS）emm分型、地域与毒力基因分布的相关性。方法 采集北京和上海地区猩红热患儿标本进行GAS分离鉴定,应用聚合酶链式反应（PCR）方法检测emm基因型以及13种毒力基因（speA、speB、speC、speF、speG、speH、speI、speJ、speK、speL、speM、ssa和smeZ）携带情况,分析GAS菌株毒力基因分布与地域、emm基因型的关系。结果 114株GAS分离株中,emm1.0型60株,emm12.0型54株;52株分离自北京（emm1.0型31株,emm12.0型21株）,62株分离自上海（emm1.0型29株,emm12.0型33株）。speA、speC、speG、speH、speI、speJ、speK、ssa和smeZ基因携带率分别为52.6％、76.3％、80.7％、43.9％、55.3％、57.0％、75.4％、97.4％、93.0％,speB和speF携带率均为98.2%,未检出speL、speM基因。北京地区emm1.0型与emm12.0 GAS菌株speA、speH、speI和speJ基因携带率比较,上海地区emm1.0型与emm12.0 GAS菌株speA、speH、speI、speJ和speK基因携带率比较,差异均有统计学意义（均P<0.05）。emm1.0型GAS菌株speC、speH、speJ和speK基因携带率北京与上海两地区比较,差异均有统计学意义（均P<0.05）,emm12.0型GAS菌株speC、speH、speI、speJ和speK基因北京与上海两地区比较,差异均有统计学意义（均P<0.05）。结论 GAS菌株毒力基因的分布状态因emm基因分型及地域不同存在差异。     

The dynamic nature of group A streptococcal epidemiology in tropical communities with high rates of rheumatic heart disease

Prospective surveillance was conducted in three remote Aboriginal communities with high rates of rheumatic heart disease in order to investigate the epidemiology of group A beta-haemolytic streptococci (GAS). At each household visit, participants were asked about sore throat. Swabs were taken from all throats and any skin sores. GAS isolates were emm sequence and pattern-typed using standard laboratory methods. There were 531 household visits; 43 different emm types and subtypes (emmST) were recovered. Four epidemiological patterns were observed. Multiple emmST were present in the population at any one time and household acquisition rates were high. Household acquisition was most commonly via 5- to 9-year-olds. Following acquisition, there was a 1 in 5 chance of secondary detection in the household. Throat detection of emmST was brief, usually <2 months. The epidemiology of GAS in these remote Aboriginal communities is a highly dynamic process characterized by emmST diversity and turnover.     

Nursing home outbreak of invasive group a streptococcal infections caused by 2 distinct strains.

OBJECTIVE: To identify factors contributing to a cluster of deaths from invasive group A streptococcus (GAS) infection in a nursing home facility and to prevent additional cases. DESIGN: Outbreak investigation. SETTING: A 146-bed nursing home facility in northern Nevada. METHODS: We defined a case as the isolation of GAS from a normally sterile site in a resident of nursing home A. To identify case patients, we reviewed resident records from nursing home A, the local hospital, and the hospital laboratory. We obtained oropharyngeal and skin lesion swabs from staff and residents to assess GAS colonization and performed emm typing on available isolates. To identify potential risk factors for transmission, we performed a cohort study and investigated concurrent illness among residents and surveyed staff regarding infection control practices. RESULTS: Six residents met the case patient definition; 3 (50%) of them died. Among invasive GAS isolates available for analysis, 2 distinct strains were identified: emm11 (3 isolates) and emm89 (2 isolates). The rate of GAS carriage was 6% among residents and 4% among staff; carriage isolates were emm89 (8 isolates), emm11 (2 isolates), and emm1 (1 isolate). Concurrently, 35 (24%) of the residents developed a respiratory illness of unknown etiology; 41% of these persons died. Twenty-one (30%) of the surveyed employees did not always wash their hands before patient contacts, and 27 (38%) did not always wash their hands between patient contacts. CONCLUSIONS: Concurrent respiratory illness likely contributed to an outbreak of invasive GAS infection from 2 strains in a highly susceptible population. This outbreak highlights the importance of appropriate infection control measures, including respiratory hygiene practices, in nursing home facilities.     

Distribution of emm genotypes among group A streptococcus isolates from patients with severe invasive streptococcal infections in Japan, 2001-2005

We surveyed emm genotypes of group A streptococcus (GAS) isolates from patients with severe invasive streptococcal infections during 2001-2005 and compared their prevalence with that of the preceding 5 years. Genotype emm1 remained dominant throughout 2001 to 2005, but the frequency rate of this type decreased compared with the earlier period. Various other emm types have appeared in recent years indicating alterations in the prevalent strains causing severe invasive streptococcal infections. The cover of the new 26-valent GAS vaccine fell from 93.5% for genotypes of isolates from 1996-2000 to 81.8% in 2001-2005.     

Distribution of emm genotypes and superantigen genes of Streptococcus pyogenes isolated in Japan, 1994-9

The purpose of this study was to examine characteristic profiles of Streptococcus pyogenes clinical isolates isolated in Japan during 1994-9. Genotyping of the M protein (emm typing) revealed that emm types 12 and 28 were the most common among 316 isolates. Most of the emm12 isolates were isolated from mucosa, while emm58 and emm89 were from skin. Moreover, the emm3 isolates were dominant in invasive infections. The distribution of 6 superantigen genes showed that all isolates harboured the mf gene and many had the speG gene. Invasive isolates were shown to have the ssa gene at a higher rate (76%) than noninvasive (37%). The distribution of superantigens was significantly different between emm types, but not between isolation sites. These results suggest that the distribution of emm types is related to isolation site, whereas superantigen distribution is related to clinical features of S. pyogenes infections.     

Outbreak of group A streptococcal throat infection: don't forget to ask about food

We report a large foodborne outbreak due to group A streptococci (GAS), which caused acute tonsillo-pharyngitis in 200-250 patrons of a company canteen in Copenhagen, Denmark, in June 2006. A retrospective cohort study of canteen users showed that consumption of cold pasta was associated with an increased risk of illness (attack rate 68%, risk ratio 4.1, P<0.0001). Indistinguishable GAS strains (emm89, T-type 3/13/B3264) were cultured from three cases and a cook, who had prepared the pasta. To our knowledge, pasta has previously only twice been incriminated as the source of a GAS outbreak. Only six foodborne GAS outbreaks have been reported in Europe since 1970, four of them in Sweden or Denmark. This geographical clustering suggests that foodborne GAS outbreaks are probably under-recognized elsewhere.     

Development of an opsonophagocytic killing assay for group a streptococcus

Group A Streptococcus (GAS) or Streptococcus pyogenes is responsible for an estimated 500,000 deaths worldwide each year. Protection against GAS infection is thought to be mediated by phagocytosis, enhanced by bacteria-specific antibody. There are no licenced GAS vaccines, despite many promising candidates in preclinical and early stage clinical development, the most advanced of which are based on the GAS M-protein. Vaccine progress has been hindered, in part, by the lack of a standardised functional assay suitable for vaccine evaluation. Current assays, developed over 50 years ago, rely on non-immune human whole blood as a source of neutrophils and complement. Variations in complement and neutrophil activity between donors result in variable data that is difficult to interpret. We have developed an opsonophagocytic killing assay (OPKA) for GAS that utilises dimethylformamide (DMF)-differentiated human promyelocytic leukemia cells (HL-60) as a source of neutrophils and baby rabbit complement, thus removing the major sources of variation in current assays. We have standardised the OPKA for several clinically relevant GAS strain types (emm1, emm6 and emm12) and have shown antibody-specific killing for each emm-type using M-protein specific rabbit antisera. Specificity was demonstrated by pre-incubation of the antisera with homologous M-protein antigens that blocked antibody-specific killing. Additional qualifications of the GAS OPKA, including the assessment of the accuracy, precision, linearity and the lower limit of quantification, were also performed. This GAS OPKA assay has the potential to provide a robust and reproducible platform to accelerate GAS vaccine development.     

Invasive group A streptococcal infection and vaccine implications, Auckland, New Zealand

We aimed to assess the effect of invasive group A streptococcal (GAS) infection and the potential effects of a multivalent GAS vaccine in New Zealand. During January 2005-December 2006, we conducted prospective population-based laboratory surveillance of Auckland residents admitted to all public hospitals with isolation of GAS from normally sterile sites. Using emm typing, we identified 225 persons with confirmed invasive GAS infection (median 53 years of age; range 0-97 years). Overall incidence was 8.1 cases per 100,00 persons per year (20.4/100,000/year for Maori and Pacific Islanders; 24.4/100,000/year for persons >65 years of age; 33/100,000/year for infants <1 year of age). Nearly half (49%) of all cases occurred in Auckland's lowest socioeconomic quintile. Twenty-two persons died, for an overall case-fatality rate of 10% (63% for toxic shock syndrome). Seventy-four percent of patients who died had an underlying condition. To the population in our study, the proposed 26-valent vaccine would provide limited benefit.     

Structure-based design of broadly protective group a streptococcal M protein-based vaccines

BackgroundA major obstacle to the development of broadly protective M protein-based group A streptococcal (GAS) vaccines is the variability within the N-terminal epitopes that evoke potent bactericidal antibodies. The concept of M type-specific protective immune responses has recently been challenged based on the observation that multivalent M protein vaccines elicited cross-reactive bactericidal antibodies against a number of non-vaccine M types of GAS. Additionally, a new “cluster-based” typing system of 175 M proteins identified a limited number of clusters containing closely related M proteins. In the current study, we used the emm cluster typing system, in combination with computational structure-based peptide modeling, as a novel approach to the design of potentially broadly protective M protein-based vaccines.MethodsM protein sequences (AA 16–50) from the E4 cluster containing 17 emm types of GAS were analyzed using de novo 3-D structure prediction tools and the resulting structures subjected to chemical diversity analysis to identify sequences that were the most representative of the 3-D physicochemical properties of the M peptides in the cluster. Five peptides that spanned the range of physicochemical attributes of all 17 peptides were used to formulate synthetic and recombinant vaccines. Rabbit antisera were assayed for antibodies that cross-reacted with E4 peptides and whole bacteria by ELISA and for bactericidal activity against all E4G AS.ResultsThe synthetic vaccine rabbit antisera reacted with all 17 E4 M peptides and demonstrated bactericidal activity against 15/17 E4G AS. A recombinant hybrid vaccine containing the same E4 peptides also elicited antibodies that cross-reacted with all E4 M peptides.ConclusionsComprehensive studies using structure-based design may result in a broadly protective M peptide vaccine that will elicit cluster-specific and emm type-specific antibody responses against the majority of clinically relevant emm types of GAS.     

New 30-valent M protein-based vaccine evokes cross-opsonic antibodies against non-vaccine serotypes of group A streptococci

Our previous studies have shown that recombinant multivalent vaccines containing amino-terminal M protein fragments from as many as 26 different serotypes of group A streptococci (GAS) evoked opsonic antibodies in animals and humans. In the present study, we constructed a new 30-valent vaccine containing M protein peptides from GAS serotypes prevalent in North America and Europe. The vaccine was immunogenic in rabbits and evoked bactericidal antibodies against all 30 vaccine serotypes of GAS. In addition, the vaccine antisera also contained significant levels of bactericidal antibodies against 24 of 40 non-vaccine serotypes of GAS. These results indicate that the potential efficacy of the new multivalent vaccine may be greater than predicted based on the “type-specific” M peptides represented.     

淄博市1956-2014年猩红热发病趋势和流行特征

目的 分析淄博市1956-2014年猩红热的发病趋势和流行特点,了解2013-2014年淄博市A群溶血性链球菌（group A streptococcus,GAS）的emm基因型,为开展防控工作提供依据。方法 收集猩红热历史疫情资料和国家疾病监测信息系统报告的猩红热病例,利用描述性流行病学方法进行分析。采集2013-2014年病例标本,培养分离GAS,应用PCR扩增emm基因,确定基因型别。结果 1956-2014年淄博市共报告猩红热病例13 470例,死亡24例,病死率0.18%。年发病率介于0.51～70.47/10万之间。59年间共出现7个流行周期,5～10年为一个周期。每年有春季（4～6月）和冬季（11～1月）2个发病高峰,15岁以下儿童占93.22%。2年分离14株GAS,共检测出6个基因亚型,emm1.0型6株、emm1.24型1株、emm1.70型1株、emm12.0型4株、emm12.7型1株、emm75.0型1株。结论 59年间淄博市猩红热疫情呈现波动式下降,发病呈现明显的周期性和季节性。2013、2014年淄博市GAS的主要流行株是emm1.0和emm12.0。小学生和托幼儿童是高危人群,应作为防控工作重点对象。     

Isolation rates of Streptococcus pyogenes in patients with acute pharyngotonsillitis and among healthy school children in Iran

We examined three populations from the Tehran region and the North part of Iran (Gilan), in all more than 5000 individuals, for carriage of Streptococcus pyogenes (group A streptococci; GAS). Children or adults with acute pharyngotonsillitis and healthy school children harboured GAS in 34-1, 20.0 and 21.0%, respectively. Typing of 421 randomly selected isolates showed a predominance of M-types M4, M5, M11, M12, as well as the provisional type 4245; however, many of the isolates were T and M non-typable. Forty-three percent of all strains were opacity factor (OF) negative. The type distribution differed markedly from that reported in 1973-4, when M types 1 and 12 were predominant.     

Group G streptococcal bacteremia in Jerusalem

Group G Streptococcus (GGS) can cause severe infections, including bacteremia. These organisms often express a surface protein homologous to the Streptococcus pyogenes M protein. We retrospectively studied the characteristics of patients from the Hadassah Medical Center with GGS bacteremia from 1989 to 2000. Ninety-four cases of GGS bacteremia were identified in 84 patients. The median age was 62 years, 54% were males, and 92% had underlying diseases (35% had a malignancy, and 35% had diabetes mellitus). The most frequent source for bacteremia was cellulitis (61%). emm typing of 56 available isolates disclosed 13 different types, including 2 novel types. Six patients had recurrent bacteremia with two to four bacteremic episodes, five had chronic lymphatic disorders, and two had emm type stG840.0 in every episode. Recurrent bacteremia has not been described for invasive group A Streptococcus. We describe an entity of recurrent GGS bacteremia, which is associated with lymphatic disorders and possibly with emm stG840.0.