Epidemiology and distribution of 10 superantigens among invasive Streptococcus pyogenes disease in Germany from 2009 to 2014

A nationwide laboratory-based surveillance study of invasive S. pyogenes infections was conducted in Germany. Invasive isolates (n = 719) were obtained between 2009 and 2014. Most isolates were obtained from blood (92.1%). The proportions of isolates from cerebrospinal fluid, pleural fluid, synovial fluid and peritoneal fluid were 3.9%, 1.8%, 1.7% and 0.6%, respectively. The most common emm types were emm 1 (31.8%), emm 28 (15.4%) and emm 89 (14.5%). The most common superantigen genes (speA, speC, speG, speH, speI, speJ, speK, speL, speM, ssa) identified from S. pyogenes were speG (92.1%), speJ (50.9%), and speC (42.0%). Significant associations of superantigen genes with underlying conditions or risks were observed in speG, speH, speJ, and speK. Significant associations between emm types or superantigen genes with clinical complications were observed in emm type 3 and in superantigen gene speA 1–3. Most frequent clinical manifestations included sepsis 59.4%, STSS 6.3%, meningitis 5.4%, and necrotizing fasciitis 5.0% (significantly associated with emm1).


Introduction
Streptococcus pyogenes (Lancefield group A streptococcus; GAS) is a major human pathogen and responsible for a wide range of both suppurative and non-suppurative diseases, e.g. pharyngitis, erysipelas, septicaemia, meningitis, pneumonia and the notably severe manifestations necrotising fasciitis (NF) and streptococcal toxic shock syndrome (STSS). Suppurative infections and post-infection sequelae, e.g. acute rheumatic fever, rheumatic heart disease and glomerulonephritis, result in substantial human morbidity [1]. Invasive infections caused by S. pyogenes (iGAS) have been reported increasingly since the mid-to late 1980s [2], and recent upsurges in iGAS infections were reported from England [3], Ireland [4,5] and Sweden [6]. The global burden of invasive S. pyogenes disease is high, and there are estimated to be at least 663,000 new cases and 163,000 deaths worldwide each year. Beyond this, there are more than a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 111 million cases of S. pyogenes pyoderma and over 616 million cases of pharyngitis annually [7]. Among the many virulence factors produced by S. pyogenes, the M protein is considered to be of major importance. The M protein is a fimbrial protein located on the cell surface. The emm gene, which encodes the M protein, is used as the basis for typing S. pyogenes. Marked changes in the distribution of emm types circulating in Europe have been noticed over the last three decades [2]. Furthermore, there seem to be huge differences concerning the global distribution of emm types. A systematic review of the global distribution of GAS emm types found the epidemiology in Africa and the Pacific region to be different from that in other regions, particularly high-income countries. In Africa and the Pacific, there were no dominant emm types and a higher diversity of emm types, and many of the emm types common in other parts of the world were less common (including emm 1, 4, 6 and 12) [8].
In this study, we analysed the superantigens speA, speC, speG, speH, speI, speJ, speK, speL, speM, and ssa for all isolates. The present investigation compares the emm types and the superantigen toxin genes of 719 invasive S. pyogenes strains collected in a nationwide voluntary laboratory-based surveillance in Germany during 2009 to 2014. Clinical manifestations, clinical complications, underlying conditions and risk factors are analysed.

Study design
German microbiological laboratories were invited to send their isolates to the German National Reference Center for Streptococci (NRCS; Aachen, Germany). In total, 719 isolates were sent by 130 laboratories located all over Germany between 2009 and 2014. Isolates were included into the study when they met the criteria of an invasive infection according to the definition of the Working Group on Severe Streptococcal Infections 1993 [19], i.e. isolation from a normally sterile site (e.g., blood, cerebrospinal fluid, synovial fluid).
In order to collect the underlying data, a detailed questionnaire was filled out for each specimen sent by the participating centers. In the few cases without enclosed questionnaires, the completion of the data sheet was requested retrospectively. The data included gender and age of the patient, the diagnoses (including certain specified diagnoses like STSS, NF, septicaemia, pneumonia, cellulitis and puerperal sepsis), and information about the clinical course (including information about presence of shock, adult respiratory distress syndrome, presence of artificial ventilation, renal failure, soft-tissue necrosis, disseminated intravascular coagulation, liver abnormality and exanthema). Possible risk factors analysed included the presence of immunosuppression, concomitant surgery, diabetes mellitus, chronic skin lesions, hospital acquired infection and intravenous drug abuse.

Statistical analysis
Continuous variables were summarized by means and corresponding standard deviations. Categorical variables were summarized by absolute and relative frequencies. Univariate logistic regression models were used for variable selection, and a selection criterion of p < 0.05 was used for inclusion into multivariate logistic regression models. For each emm type and each superantigen, we investigated the possible influence on each outcome parameter (all diagnoses and all clinical complications). In contrast, the influence of all risk factors was investigated for all emm types and all superantigens (here the outcome parameter). Influence factors with a p value of p < 0.05 as well as age and sex were selected for the corresponding multivariate models. Univariate models were also constructed to examine possible relationships between emm type and superantigen genes. For sex, odds ratios >1 correspond to relationships which occur more commonly in males, while odds ratios <1 occur more commonly in females. For age, odds ratios >1 correspond to a relationship with increasing age, and odds ratios <1 correspond to decreasing age. Only outcome variables with 15 or more events are analysed in regression models [21]. Further possible estimation problems are described in the discussion section. All tests were two-sided and assessed at the 5% significance level. Because of the exploratory nature of the study we made no adjustment to the significance level of the several multivariate models. Statistical analyses were performed using R software, version 3.3.2. The complete data set used for the analyses is included as a supplementary table, S1 Table. Ethical statement An ethical approval or patients' consent was not required since the study only includes microbiological samples sent to the German National Reference Center for Streptococci on an anonymized basis by the sending microbiological laboratories, and did not involve human subjects or material.  (13), synovial fluid (12), and peritoneal fluid (4). A seasonal variation was noted, with most cases reported in winter and early spring (Fig 1).

Results
The age-specific incidence of iGAS infections is shown in Fig 2. For the 719 patients, the mean age was 53.5 years, the median 59 years (range 0-97 years). A higher amount of cases per 100,000 inhabitants in the respective age groups was found in children up to 5 years and in adults ! 60 years. Among the latter, in our study the incidence was relatively constant among adults in the age groups from 30 to 59 years, but rose with every decade of age among those aged ! 60 years. For adults from 70-79 years, and especially those aged ! 80 years, the incidence even exceeded those among children from 0-5 years of age.
The correlations between risks/underlying conditions, diagnoses and clinical complications and emm types or superantigens found in the statistical analysis are shown in Tables 4, 5 and 6. Among underlying conditions and risk factors, speH, speJ, and speK were significantly associated with chronic skin lesions, and speG was significantly associated with diabetes. Among clinical complications, emm 1 was non-significantly associated with hypotensive shock, DIC, renal insufficiency, liver abnormality, soft tissue necrosis, and exanthema. Hypotensive shock, DIC, and renal insufficiency were non-significantly associated with emm 28. Superantigen speC was non-significantly associated with DIC, renal insufficiency, and exanthema; speA 1-3 with hypotensive shock, DIC, renal insufficiency, liver abnormality (significantly), soft tissue necrosis, and exanthema. Superantigen speJ was non-significantly associated with hypotensive shock and exanthema; speM was non-significantly associated with soft tissue necrosis. In meningitis cases, emm types 1 and 89, as well as speA 1-3 were predictors. emm 1, speC, and speJ were all non-significantly associated with NF (emm1 reached statistical significance for NF) and sepsis. Septic arthritis was significantly associated with emm 28.
Additionally, we established some correlations between age, sex, and emm types and superantigen genes.Superantigen speH was associated with decreasing age. Among clinical complications, coagulopathy, respiratory distress, and exanthema were significantly associated with decreasing age, while renal insufficiency was significantly associated with increasing age. Decreasing age was associated with speH and speJ as predictors of chronic skin lesions. And in the studied diagnoses, meningitis was associated with decreasing age.

Discussion
In this paper we present the results of 6 years of surveillance of iGAS disease in Germany.
Reported iGAS cases in Germany are low in comparison with surveillance programs from other countries. This might, at least in part, be explained by the voluntary nature of the German surveillance system, resulting in a smaller number of cases being referred to the reference isolates. 75 cases had one underlying condition, 18 cases had two underlying conditions, and two cases had three underlying conditions. a NSAID, nonsteroidal anti-inflammatory drugs.    laboratory and a potential underreporting of invasive S. pyogenes infections. In comparison with previous German surveillance periods, the incidence per year is slightly, but not significantly, higher in the current study (0.15 cases/100,000 individuals) than in previous surveillance periods (1996-2002, 0.1 cases [9]; 2003-2007, 0.13 cases [22]). The seasonal occurrence   of iGAS disease with most cases reported in winter and early spring is congruent with the patterns observed in other countries [2]. In the present study, emm 1 was the most prevalent emm type, which is consistent with results from the USA [23], Australia [24], Japan [25], and across Europe [2,9,11], followed in frequency by the emm types 28, 89, 3 and 12. These five emm types are responsible for over three-fourths of iGAS disease in the current German surveillance period and these emm types have been reported to be among the most prevalent in the United States [23], Denmark [26,27], and other European countries [11] as well. Compared to the two previous surveillance periods in Germany (1996Germany ( -2002 and 2003-2007 [22]), overall there seems to be no  [26,28]. While our models are exploratory in nature, some underlying conditions are nevertheless clear risk factors for iGAS disease. Diabetes is a risk factor for infection with strains harbouring speG. Chronic skin lesions are a risk factor for infection with strains harboring speH, speJ, and speK. Among the studied clinical complications, significant associations were found only with speA 1-3 (with liver abnormality), and emm 3, with respiratory distress. Among the studied diagnoses, significant associations were found with emm type 1 (with NF), and emm type 28 (with septic arthritis).
The relevance of erythrogenic toxin-and superantigen genes relating to invasive infections remains inconclusive, despite extensive literature on this topic [17], particularly since they are also common in non-invasive isolates. In our study, even the statistically significant results did not result in odds ratios far above or below one. Most emm types were characterized by the presence of one or two specific toxin gene profiles [29,30]. Hypothetically, at least one toxin gene is required in order for severe GAS disease to manifest [27]. Indeed, in our study, only 10 of 719 cases (1.4%) did not have any of the superantigen genes we examined, of which two cases were from patients with a co-occurring serious illness (diabetes). There are no clear statistical relationships between diagnosis or clinical complications and the samples without any detected superantigens. Samples without any of the studied superantigens were from only five emm types, emm77 (n = 5), emm60 (n = 2), emm3 (n = 1), emm63 (n = 1), and emm165 (n = 1). Since we did not examine superantigen smeZ, we cannot rule out the possibility that these ten samples harbour this superantigen. Further research is necessary to elucidate the interrelation between superantigen gene combinations, emm types and disease pattern of iGAS infections.
Supporting information S1