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Background: The incidence and severity of invasive group A streptococcal infection demonstrate great variability over time, which at least, in part, seems to be related to group A streptococcal type distribution among the human population., Methods: An enhanced surveillance study of invasive group A streptococcal infection (746 isolates) was performed in Sweden from April 2002 through December 2004. Noninvasive isolates from either the throat or skin (773 isolates) were collected in parallel for comparison. Clinical and epidemiological data were obtained from 88% of patients with invasive disease and were related to isolate characteristics, including T type, emm sequence type, and the presence of 9 superantigen genes, as well as pulsed-field gel electrophoresis pattern comparisons of selected isolates., Results: The annual incidence was 3.0 cases per 100,000 population. Among the patients with invasive disease, 11% developed streptococcal toxic shock syndrome, and 9.5% developed necrotizing fasciitis. The overall case-fatality rate was 14.5%, and 39% of the patients with streptococcal toxic shock syndrome died (P<.001). The T3/13/B3264 cluster accounted for 33% of invasive and 25% of noninvasive isolates. Among this most prevalent type cluster, emm types 89 and 81 dominated. Combined results from pulsed-field gel electrophoresis, emm typing, and superantigen gene profiling identified subgroups within specific emm types that are significantly more prone to cause invasive disease than were other isolates of the same type., Conclusions: This study revealed a changing epidemiology of invasive group A streptococcal infection in Sweden, with emergence of new emm types that were previously not described. The results also suggest that some clones may be particularly prone to cause invasive disease.

The immune response against the infecting group A streptococcus (GAS) extracellular products (EP) was determined in acute- and convalescent-phase sera from 75 patients with different clinical manifestations of GAS infection. All EP elicited a high proliferative response in human peripheral blood mononuclear cells. In patients with bacteremia, low neutralization in acute-phase sera was associated with development of streptococcal toxic shock syndrome. Lack of neutralization in acute-phase sera was more common in patients infected with the T1emm1 serotype. The majority of patients did not develop the ability to neutralize the mitogenic activity of their infecting isolate despite a significant increase in enzyme-linked immunosorbent assay titer in early convalescent-phase sera. In patients with the ability to neutralize GAS EP, the immune response remained high over at least 3 years. In contrast, the neutralization capacity conferred by intravenous immunoglobulin and/or plasma treatment disappeared within 3 months.

Surveillance of group A streptococcus (GAS) infections in Sweden during 1996-1997 indicated that T28 isolates were dominant, whereas T1M1 infections were uncommon. Circulating T28 isolates were nearly all emm28, MLST52, and these clones had also been prevalent 10 years earlier. Isolates from invasive and noninvasive infections were of similar types and prevalences. The average national incidence of invasive episodes was 2.9/100,000 population but varied between 0 and 8.3/100,000 population in different counties. It increased markedly with age, reaching 22.9 episodes/100,000 among people aged > or =90 years. The incidence of puerperal sepsis was higher than expected (22.4/100,000 of those at risk), with 1 death. Overall mortality was 16% and was associated with preexisting chronic disease (P=.002). Streptococcal toxic shock syndrome (STSS) developed in approximately 15% of patients with invasive episodes, with a mortality rate of 45%. The use of nonsteroidal anti-inflammatory drugs was not found to be associated with the development of STSS.

The present study shows that active, self-splicing group II intron GBSi1 is located downstream of the C5a-peptidase gene, scpB, in some group B streptococcus (GBS) isolates that lack insertion sequence IS1548. IS1548 was previously reported to be often present at the scpB locus in GBS isolated in association with endocarditis. Since none of 67 GBS isolates examined, 40 of which were of serotype III, harbored both IS1548 and GBSi1, these two elements are suggested to be markers for different genetic lineages in GBS serotype III. The DNA region downstream of scpB in GBS isolates harboring either GBSi1, IS1548, or none of these mobile elements was found to encode the laminin binding protein, Lmb, which shows sequence similarities to a family of streptococcal adhesins. IS1548 is inserted 9 bp upstream of the putative promoter for lmb, while the insertion site for GBSi1 is located 88 bp further upstream. Sequences highly similar to GBSi1 exist also in Streptococcus pneumoniae. An inverted repeat sequence, with features typical of transcription terminators, was identified immediately upstream of the insertion site for the group II intron both in the GBS and S. pneumoniae sequences. This motif is suggested to constitute a target for the GBS intron as well as for rather closely related introns in Bacillus halodurans, Pseudomonas alcaligenes, and Pseudomonas putida. When transcripts containing the GBSi1 intron were incubated at high concentrations of ammonium and magnesium, a major product with the expected length and sequence for the ligated exons was generated. Unlike, however, all members of group II investigated so far, the excised intron was in linear, rather than in a branched (lariat), form.

A chemiluminescence method was used to study opsonization of group A Streptococcus (GAS) of serotype T1M1 in serum samples ("sera") obtained from Swedish patients with invasive and noninvasive GAS infection and from healthy blood donors. Acute-phase serum samples ("acute sera") generally demonstrated low ability to opsonize the patient's own GAS isolate, regardless of clinical manifestation. Only approximately 15% of serum samples obtained from healthy blood donors demonstrated high opsonic activity against a standard T1M1 strain. Opsonization of 62 T1M1 isolates (obtained during 1980-1998) by a single immune serum sample showed considerable variation; this indicates that high opsonic immunity may develop only against the infecting isolate or identical clones. T1M1 GAS isolated from 1987 through 1990 were better opsonized by the immune serum sample than were isolates obtained before 1987 or after 1990, a finding that suggests a temporal change of the surface properties that affect opsonization.

Forty group B Streptococcus (GBS) isolates obtained from Europe and the United States previously reported to be nontypeable (NT) by capsule serotype determination were subjected to buoyant density gradient centrifugation. From nearly half of the isolates capsule-expressing variants could be selected. For characterization of the remaining NT-GBS isolates, the capsule operon (cps) was amplified by the long-fragment PCR technique and compared by restriction fragment length polymorphism (RFLP) analysis. The patterns from serotype reference isolates (n = 32) were first determined and used as a comparison matrix for the NT-GBS isolates. Using two restriction enzymes, SduI and AvaII, cluster analysis revealed a high degree of similarity within serotypes but less than 88% similarity between serotypes. However, serotypes III and VII were each split in two distant RFLP clusters, which were designated III(1) and III(2) and VII(1) and VII(2), respectively. Among the isolates that remained NT after repeated Percoll gradient selections, two insertional mutants were revealed. Both were found in blood isolates and harbored insertion sequence (IS) elements within cpsD: one harbored IS1548, and the other harbored IS861. All other NT-GBS isolates could, by cluster analysis, be referred to different serotypes by comparison to the RFLP reference matrix. In pulsed-field gel electrophoresis of SmaI-restricted chromosomal DNA, patterns from allelic type 1 and 2 isolates were essentially distributed in separate clusters in serotypes III and VII. A covariation with insertion sequence IS1548 in the hylB gene was suggested for serotype III, since allelic type III(1) harboring IS1548 in hylB, clustered separately. The variation in serotype VII was not dependent on the presence of IS1548, which was not detected at any position in the type VII chromosome.

To investigate the role of allelic variants of streptokinase in the pathogenesis of acute poststreptococcal glomerulonephritis (APSGN), site-specific integration plasmids were constructed, which contained either the non-nephritis-associated streptokinase gene (skc5) from the group C streptococcal strain Streptococcus equisimilis H46A or the nephritis-associated streptokinase gene (ska1) from the group A streptococcal nephritogenic strain NZ131. The plasmids were introduced by electroporation and homologous recombination into the chromosome of an isogenic derivative of strain NZ131, in which the streptokinase gene had been deleted and which had thereby lost its nephritogenic capacity in a mouse model of APSGN. The introduction of a non-nephritis-associated allelic variant of streptokinase did not rescue the nephritogenic capacity of the strain. The mutant and the wild-type strains produced equivalent amounts of streptokinase. Complementation of the ska deletion derivative with the original ska allele reconstituted the nephritogenicity of wild-type NZ131. The findings support the hypothesis that the role of streptokinase in the pathogenesis of APSGN is related to the allelic variant of the protein.

Analysis of 132 group A streptococcal (GAS) isolates from 151 invasive episodes, including streptococcal toxic shock syndrome (STSS), from 1983 to 1995 showed great genetic variation by use of T serotyping in combination with restriction fragment length polymorphism. In contrast, genetically homogenous T1M1 isolates appeared in epidemic patterns with significantly increased risk of STSS. The speA gene, with the allelic variants speA2 and speA3 carried by the T1M1 and T3M3 serotypes, respectively, was strongly associated with STSS. Infection with a GAS isolate carrying speA, alcohol abuse, and malignancy recently treated with cytostatic drugs were factors independently related to STSS. Neutralization of SpeA lymphocyte mitogenicity was totally absent in sera from patients with STSS and low in sera from persons with uncomplicated bacteremia compared with levels in sera from uncomplicated erysipelas. Neutralization of SpeB was significantly lower in sera of patients with STSS than in sera from persons with bacteremia or erysipelas.

Considerable knowledge has been accumulated regarding the characteristics of acute post-streptococcal glomerulonephritis (APSGN), and many attempts have been made to identify a streptococcal factor or factors responsible for triggering this disease. However, the pathogenic mechanism behind APSGN remains largely unknown. As glomerular deposition of C3 is generally demonstrated before that of IgG in the disease process, it is likely that the inflammatory response is initiated by renal deposition of a streptococcal product, rather than by deposition of antibodies or pre-formed immune complexes. During recent years, a number of streptococcal products have been suggested to be involved in the pathogenic process. In this review, possible roles of these factors are discussed in the context of the clinical and renal findings most often demonstrated in patients with APSGN. Streptokinase was observed to be required in order to induce signs of APSGN in mice, and a number of findings suggest that the initiation of the disease may occur as a result of renal binding by certain nephritis-associated variants of this protein. However, additional factors may be required for the development of the disease.

In a retrospective study of invasive infections due to group A Streptococcus (GAS) in Stockholm during 1987 to 1995, the average incidence per 100,000 residents per year was 2.3, varying between 3.7 per 100,000 (in 1988) and 1.3 per 100,000 (in 1993). Incidence was 1.8 in the age group of 0-4 years but otherwise increased by age, from 0.48 in the age group of 5-14 years to 6.1 among those over 65 years of age. A review of 151 invasive episodes occurring in 1983-1995 showed cyclic increases of infections due to T1M1-serotype strains during 1986-1990 and 1993-1995. The T1M1 serotype accounted for 27 (20%) of 135 available GAS strains. Streptococcal toxic shock syndrome (STSS) developed in 19 (13%) of the 151 episodes. The case fatality rate was 11% overall but 47% among patients with STSS. In a multivariate logistic regression model, STSS was associated with a history of alcohol abuse (odds ratio [OR], 6.3; P = .004) and infection with a T1M1 strain (OR, 6.7; P = .007). Case fatality was associated with age (OR, 14.5; P = .08), immunosuppression (OR, 4.7; P = .02), and STSS (OR, 21.5; P < .0001) but not with T1M1 infection. Hypotension was significantly associated with a fatal outcome, regardless of whether STSS developed (P < .0001).

Hyaluronidase has been postulated to be a virulence factor in group B streptococci (GBS). No hyaluronidase activity was found in 15 of 50 GBS isolates from adults studied. Most of these hyaluronidase-negative strains belonged to serotype III. In strains lacking hyaluronidase activity, an insertion of 1317 nucleotides was found in the hyaluronidase gene. The fragment was cloned and sequenced and found to have characteristics of a novel insertion sequence, designated IS1548. As well as in GBS serotype III, this sequence was found in 3 of 6 serotype II isolates and in all 10 group A streptococcal strains (GAS) tested. Homologies were found with repeated sequences in Streptococcus pneumoniae and with H repeats in Escherichia coli. All GBS strains harboring IS1548 and some GAS strains had one copy of IS1548 located downstream of the C5a peptidase gene. IS1548 was present in 9 of 13 GBS isolates from blood in endocarditis patients and in 3 of 22 vaginally colonizing strains.

Group A streptococcal infections are sometimes followed by the inflammatory kidney disease acute post-streptococcal glomerulonephritis (APSGN). To test the importance of streptokinase in the pathogenesis of this disease, isogenic strains of the nephritis isolate NZ131, differing only in the ability to produce streptokinase of the nephritis-associated ska1 genotype, were used for infection in a mouse tissue cage model for APSGN. Streptokinase production was found to be a prerequisite for the capacity of the strain to induce APSGN in mice. In addition, streptokinase was demonstrated in the kidneys of mice infected with the nephritogenic NZ131 and EF514 strains. After infection with the nonnephritogenic strain S84, neither streptokinase nor C3 deposition were observed. Deposition of streptokinase in the glomeruli was detected as soon as 4 days after infection. These findings provide support for the hypothesis that streptokinase initiates the nephritis process by glomerular deposition, which leads to local activation of the complement cascade. Detection of streptokinase in kidney tissue increased with the degree of glomerular hypercellularity. Thus, the severity of the pathological process may be a reflection of the degree of streptokinase deposition.

The intrafamilial spread and recurrence of group A beta-haemolytic streptococci (GAS) infections was investigated. The evaluation was based on 114 patients and their families treated with penicillin for 5 days and followed for 1 month. GAS of the same T-type as that of the isolate from the index case were found in other family members in 33% of the families. Genetic finger-printing using RFLP was performed on 33 of the isolates. The mothers dominated among the index cases, 40 patients experienced recurrences, 27 of them were clinical 28 recurrences occurred within 10 days after the end of treatment. Of 20 T-typed patients with early clinical treatment failures, infected family members were detected in 16 families (p < 0.001). In 19% of the patients GAS could be isolated from the nose. These patients had more ill family members than did other patients. An extensive intrafamilial streptococcal spread was found. Most recurrences of GAS pharyngotonsillitis after penicillin treatment are probably due to "ping pong" infection from family members.

A number of factors have been implicated in the pathogenesis of acute poststreptococcal glomerulonephritis (APSGN). The lack of a reliable animal model has made it difficult to further examine the role of these factors in the pathogenetic process. In this report, we present a tissue cage model in mice for the study of APSGN. Morphological and immunohistological changes in the kidney, resembling those of APSGN in man, were induced at high frequency in the experimental model after infection with group A streptococcal nephritis isolates. Nephritis-associated strain induced hypercellularity, occlusion of capillaries, and C3 deposition at high frequencies compared to the changes induced in animals infected with a non-nephritis-associated strain and non-infected controls. In animals infected with a nephritis isolate, hematuria and proteinuria were also detected. If penicillin treatment was initiated on the third day of infection, the development of the nephritis process was prevented. Streptokinase, as well as preabsorbing antigen and streptococcal pyrogenic exotoxin B (SpeB), have been implicated in the pathogenesis of APSGN. These proteins, as well as SpeA and SpeF, were detected in the fluids of the infectious focus, regardless of the origin of the strains and whether or not glomerulonephritis was seen. Antibodies to streptokinase were evoked in the majority of the infected animals. This immune response did not correlate with the nephritic process since hypercellularity was also seen in animals which lacked detectable streptokinase antibodies. The results show that the mouse tissue cage model can be used to study APSGN and to evaluate factors involved in the pathogenesis of the disease.

The multiorgan failure syndrome caused by group A streptococci (GAS) designated streptococcal toxic shock syndrome (STSS) is believed to be mediated by cytokines induced by superantigens. In order to study the relationship between superantigen production, cytokine levels in patient sera, and clinical GAS manifestation we examined acute-phase sera and strains from 25 patients with GAS bacteremia. The patients had various disease manifestations, including STSS (44%), erysipelas (28%), septicemia (24%), wound infections (16%), and pneumonia (12%). Serotype T1M1 dominated, representing 56% of the isolates, but also strains of other serotypes were identified. The strains were found to produce the streptococcal pyrogenic exotoxins (Spe) A, B, and F, as determined by immuno-blot analyses. There was no difference in amounts of toxin produced between strains isolated from patients with different manifestations of disease. Levels of TNF alpha, IL1 alpha, IL6, IL8, and IFN gamma in acute-phase sera were determined by use of ELISA and RIA assays. The analyses showed higher levels of IL6 in sera from patients with STSS than in sera from patients with bacteremia without shock. TNF alpha was elevated in sera from patients with STSS, as compared to sera from patients with uncomplicated pharyngotonsillitis. No increase in the levels of IL1 alpha, IL8, and IFN gamma could be found in the patient sera and there was no difference in the level of those cytokines between the various patient categories.

The proliferative response of lymphocytes induced by a new streptococcal mitogenic factor (MF) and the streptococcal pyrogenic exotoxins (Spe) A and B was determined in sera from 6 healthy persons. Responses were compared to those obtained from reference serum that lacked mitogen-specific ELISA antibodies. The sera showed individual variations in the levels of MF- and Spe-specific antibodies, as determined by ELISA. The experiments showed that most human sera, which contained mitogen-specific antibodies, could neutralize the mitogenicity of the proteins. However, there were sera that contained mitogen-specific antibodies but that did not inhibit the toxin-induced proliferation. Thus, the ELISA antibody titer did not always equal the neutralizing capacity. Sera from 27 patients with group A streptococcal bacteremia had significantly lower neutralizing ability against MF and SpeB than did sera from 25 uncomplicated tonsillitis cases.

Certain variants of streptokinase from group A streptococci have been associated with acute post-streptococcal glomerulonephritis (APSGN). The streptokinase gene (ska) has previously been grouped into nine different polymorphic genotypes of which ska1, ska2, ska6, and ska9 were identified in group A streptococci associated with clinical and experimental APSGN. A total of 53 group A streptococci isolated from Ethiopian children: five from acute rheumatic fever, 18 from APSGN, ten each from tonsillitis, impetigo and healthy carriers, were analyzed for ska gene polymorphism using polymerase chain reaction (PCR) and restriction enzyme analysis. The frequency of the nephritis-associated streptokinase genotypes was 83% among the APSGN isolates and 74% in the non-ASPGN isolates. ska2 was the most commonly found genotype with a frequency of 64% among all isolates, 66% among the APSGN isolates, and 63% among the non-APSGN isolates. ska1 was identified in 13% among all isolates and 17% among the APSGN isolates. Seventeen non-APSGN isolates from Scandinavian countries were studied for comparison and all carried either ska1 or ska2. The other nephritis-associated ska6 and ska9 were not detected among the 53 Ethiopian isolates. ska1 was exclusively associated with serum opacity reaction (SOR) producers. ska2 was evenly distributed among SOR-positive and SOR-negative isolates. The other genotypes were detected only among SOR-negative strains. The findings of the present study showed an even distribution of the nephritis-associated streptokinase gene among group A streptococcal isolates with no correlation to disease pattern. Thus additional factors must also be operative in the pathogenesis of APSGN.

Genetic diversity was found at high frequency downstream of the emm1 gene among T1M1 group A streptococci (GAS) isolated in Scandinavia during a recent epidemic. Clonal variation was also seen in the speA and speB genes but at much lower frequency; no variation was detected in the speC gene. Erythrogenic toxin A was found to be expressed at low levels in all strains; erythrogenic toxins B and C were produced in high amounts. All strains were found to harbor the speA, speB, and speC genes, regardless of the amount of toxin produced. No correlation was found between one specific T1M1 clone and the more serious infections when isolates from bacteremic patients (fatalities or survivors), those with uncomplicated infections, and healthy carriers were compared. Similar results were obtained in a family study in which 3 family members were found to be asymptomatic carriers of the same GAS T1M1 clone as in the bacteremic patient, defined by genotypic and phenotypic experiments.

A nontypeable blood isolate of group B streptococci (GBS) from a patient with endocarditis is suggested to be the nonencapsulated phase of a GBS strain, type III. From the original high-density isolate, a low-density, encapsulated phase was selected by Percoll gradient centrifugation. This phenomenon should be considered before a GBS strain is classified as truly nontypeable.

Serotypes of serious, sometimes fatal, streptococcal infections in Sweden during 1988-1989 were analyzed. The T1M1 type totally dominated, representing almost 70% of all group A streptococci from serious and uncomplicated infections at the peak of the outbreak. Immunoblots of isolates from various patient groups showed that all isolates produced high amounts of erythrogenic toxin (ET) B and high amounts of ET-C, whereas ET-A was released only in small amounts and from few isolates. ELISAs showed high antibody levels to these toxins and to the M1 antigen in patients with uncomplicated infections. Low antibody levels against M1 were seen in patients with bacteremia and in fatal cases; the latter also had low antibody levels against ET-B. It seems likely that a combination of production of large amounts of toxin and low antibody titers to it and to the M antigen of the infecting isolate are determining factors for the outcome of the infection. No signs of primary immune deficiency were noted.