Pivotal Role of Preexisting Pathogen-Specific Antibodies in the Development of Necrotizing Soft-Tissue Infections

Pivotal Role of Preexisting Pathogen-Specific Antibodies in the Development of Necrotizing... Abstract Background Necrotizing soft-tissue infections (NSTI) are the most severe form of bacterial-induced tissue pathology. Their unpredictable onset and rapid development into life-threatening conditions considerably complicate patient treatment. Understanding the risk factors for NSTI in individual patients is necessary for selecting the appropriate therapeutic option. Methods We investigated the role of pathogen-specific antibodies in the manifestation of NSTI by performing a comparative serologic approach, using plasma samples and bacterial isolates from patients with clinical NSTIs or nonnecrotizing STIs caused by Streptococcus pyogenes. We also evaluated the potential beneficial effect of intravenous immunoglobulin (IVIG) treatment. Results We identified a hitherto overlooked state of serologic susceptibility in patients with NSTIs during the earliest stages of the infection that is potentially linked to disease progression. Thus, all patients with NSTIs included in this study exhibited a deficiency in specific antibodies directed against the causative S. pyogenes strains and the majority of their exotoxins during the initial stage of the infection. We also showed that the clinical use of IVIG during the course of infection compensates the observed antibody deficiency but is unable to halt the disease progression, once tissue necrosis has developed. Conclusion These observations emphasize the requirement of preexisting pathogen-specific antibodies to prevent the irreversible progression of tissue infections into severely spreading NSTIs and urge further investigations on the beneficial effect of IVIG-based early phase intervention strategies to prevent the severe effects of this devastating bacterial infection. Antibody titers, exotoxins, intravenous immunoglobulins (IVIG), necrotizing Soft tissue infections, Streptococcus pyogenes Necrotizing soft-tissue infections (NSTIs) are the most dramatic form of bacterial-induced tissue pathology that is associated with devastating side effects, ranging from severe soft-tissue necrosis to systemic inflammation and toxic shock [1–4]. Streptococcus pyogenes is a major cause of NSTIs [5, 6]. Owing to the rapidly progressing nature of NSTI, prompt diagnosis and treatment, including aggressive surgery and antibiotics, along with possibly adjunctive therapies, such as intravenous immunoglobulin (IVIG), are critical. Limited knowledge on the abrupt onset of NSTI and on the mechanisms driving a self-limiting local tissue infection into a severely spreading necrotic stage remain a major challenge for establishing tailored strategies that interfere with NSTI development in patients at risk. Soft-tissue infections are generally initiated after a pathogen enters the hypodermal tissue through a wound or by the hematogenic spread into tissue damaged by minor trauma [7, 8]. However, the comparatively low number of tissue infections that develop into NSTI implies that the immune system is, in most instances, able to control the invading pathogen, thereby preventing the progression to NSTI. Based on these premises and on the important role of opsonic and toxin-neutralizing antibodies for controlling S. pyogenes infections [9], it can be hypothesized that serologic susceptibility characterized by a lack or low levels of specific antibodies against the pathogen and its toxins may predispose an individual to NSTI. The current study investigates the role of specific antibodies against S. pyogenes and its toxins in the development of NSTI and characterizes the impact of IVIG as adjunctive therapy in the progression of NSTI caused by S. pyogenes. This study is the first to demonstrate the protective effect of preexisting antibody titers against streptococcal surface and secreted exotoxins in preventing the irreversible progression of mild tissue infections into severe NSTI and the potential benefit of IVIG for the treatment of patients at risk. MATERIAL AND METHODS Patient Plasma Samples Patients with clinical NSTIs or nonnecrotizing STIs were recruited, and the corresponding bacterial isolates were collected within the framework of the European Union–funded project INFECT (available at: http://www.fp7infect.eu; Supplementary Table 1). Plasma samples from patients were collected when the patient was recruited in the study (day 0) and after 3 days of treatment (day 3). Written informed consent was obtained from all patients or their next of kin as soon as possible. The study is part of INFECT and is registered at ClinicalTrials.gov (NCT01790698). All samples were collected in accordance with the Declaration of Helsinki and with the approval of the regional Ethical Review Board at the Karolinska Institutet in Stockholm, Sweden (ethics permits: 2012/2110-31/2); the National Committee on Health Research Ethics in Copenhagen, Denmark (Ethics permits: 1151739); or the regional Ethical Review Board in Gothenburg, Sweden (ethics permits: 930-12) or Bergen, Norway (2012/2227/REC West). Fresh human blood samples were collected in house from the right median cubital vein of healthy middle-aged volunteers under ethical approval number BO/07/2013. Bacteria Streptococcal isolates were cultivated in Todd Hewitt broth containing 0.5% yeast extract at 37°C. Exponentially growing cultures were used (absorbance at 600 nm = 0.4) unless stated otherwise. Escherichia coli strains were cultivated in Luria-Bertani medium supplemented with appropriate concentrations of antibiotics (Supplementary Table 2) at 37°C (or 30°C after IPTG induction) horizontally shaking at 150 rpm. Clinical isolates were identified by 16S ribosomal DNA sequencing and typed for their hemolytic behavior, surface antigen presentation, and emm gene. Genomic DNA was extracted from overnight cultures and screened for the presence of 14 streptococcal exotoxin encoding genes (speA, speC, speG, speH, speI, speJ, speK, speL, speM, ssa, smeZ, speB, sic, and mac) by multiplex polymerase chain reaction (PCR) analysis as described previously [10]. All PCR products were sequenced using terminator cycle sequencing (ABI Prism). Opsonophagocytosis Assay In vitro opsonophagocytosis assays were performed using freshly isolated human granulocytes and plasma samples as previously described (Supplementary Methods). Antibody Detection and Inhibition Assays Antigen-specific immunoglobulin G (IgG) titers in patient plasma samples were determined using standard enzyme-linked immunosorbent assay (ELISA) protocols after cloning and purification of 14 streptococcal exotoxins (Supplementary Methods). SpeB (Genovis), Mac (Genovis), streptolysin O (SLO; Sigma), and hyaluronidase (Hyl; Sigma) were purchased from commercial suppliers. To characterize the neutralization of toxin effects by antibodies in IVIG or patient plasma, inhibition assays were performed. The activity of superantigens was measured using T-cell proliferation assays (Supplementary Methods). The activities of SpeB, SLO, and Sic were measured through cleavage of azocasein, red blood cell lysis, and LL-37 inhibition as previously described [11–13]. Nucleotide Sequence Accession Numbers The 16S ribosomal DNA and emm gene sequences reported in this study have been deposited in GenBank under accession numbers MF480476-MF480523 and MF538589-MF538623. Statistical Analysis Comparisons between groups were made using Student t test or by analysis of variance. P values of < .05 were considered significant. RESULTS Patients With NSTIs Lack Antibodies Against the Corresponding S. pyogenes Isolate and Its Exotoxins A cohort of 51 NSTI cases recruited within the frame of the European Union–funded project INFECT and identified as monoinfections caused by members of the genus Streptococcus were included in this study (Figure 1A). S. pyogenes was identified as the most frequent causative pathogen (63% of cases; Figure 1A). The S. pyogenes isolates comprised 11 serotypes, with emm1 being the most abundant (47%; Figure 1B). Figure 1. View largeDownload slide Species and serotype distribution of isolates from 51 necrotizing soft-tissue monoinfections caused by members of the genus Streptococcus. A, Streptococcal species distribution identified by 16S ribosomal RNA gene sequencing. B, Serotypes distribution of all 32 S. pyogenes isolates, determined by emm sequencing. Figure 1. View largeDownload slide Species and serotype distribution of isolates from 51 necrotizing soft-tissue monoinfections caused by members of the genus Streptococcus. A, Streptococcal species distribution identified by 16S ribosomal RNA gene sequencing. B, Serotypes distribution of all 32 S. pyogenes isolates, determined by emm sequencing. To investigate a potential correlation between the immunologic status of the patients and the development of NSTI, a comprehensive serologic study involving the characterization of the opsonophagocytic capacity of plasma collected at different times of infection against the corresponding bacterial isolate was performed in a representative set of 7 IVIG-treated and 7 non–IVIG-treated NSTI cases reflecting the serotype distribution of the complete set of S. pyogenes isolates. Two cases of nonnecrotizing STIs were included as study controls (Table 1). Whereas plasma samples collected on the day of study recruitment (day 0) from all 14 NSTI cases were inefficient at inducing opsonophagocytic killing of the corresponding S. pyogenes strain, plasma specimens from the 2 patients with nonnecrotizing STIs exhibited a remarkable capacity to induce opsonophagocytic elimination of S. pyogenes (Figure 2A). These findings suggest that the lack of opsonizing antibodies in patients with NSTIs may be a decisive factor for the progression from STI to NSTI. Interestingly, plasma samples collected on day 3 from all patients with NSTIs exhibited a significant increase in opsonophagocytosis activity that was more pronounced in the IVIG-treated group (Figure 2A). These observations demonstrated that opsonic antibodies are elicited in all patients with the progression of S. pyogenes infection and that IVIG treatment enhanced opsonophagocytosis of S. pyogenes, an effect that was most probably mediated by the presence of high levels of opsonic antibodies in the IVIG preparations. The presence of opsonic antibodies in IVIG preparations was demonstrated by the significant increase in bacterial killing observed after incubating the different S. pyogenes isolates with neutrophils in the presence of IVIG preparations (Figure 2B). Table 1. Cases of Streptococcus pyogenes Monoinfection Selected for the Serologic Study Isolate NSTI IVIG Treatment emm Type 2001 Yes Yes emm1 2006 Yes Yes emm1 2017 Yes Yes emm28 5003 Yes Yes emm77 5004 Yes Yes emm28 5006 Yes Yes emm1 6013 Yes Yes emm1 3005 Yes No emm89 3012 Yes No emm77 6016 Yes No emm1 6018 Yes No emm1 6025 Yes No emm1 6026 Yes No emm4 6033 Yes No emm63 6028 No No emm4 6040 No No emm28 Isolate NSTI IVIG Treatment emm Type 2001 Yes Yes emm1 2006 Yes Yes emm1 2017 Yes Yes emm28 5003 Yes Yes emm77 5004 Yes Yes emm28 5006 Yes Yes emm1 6013 Yes Yes emm1 3005 Yes No emm89 3012 Yes No emm77 6016 Yes No emm1 6018 Yes No emm1 6025 Yes No emm1 6026 Yes No emm4 6033 Yes No emm63 6028 No No emm4 6040 No No emm28 Abbreviations: IVIG, intravenous immunoglobulin; NSTI, necrotizing soft-tissue skin infection. View Large Table 1. Cases of Streptococcus pyogenes Monoinfection Selected for the Serologic Study Isolate NSTI IVIG Treatment emm Type 2001 Yes Yes emm1 2006 Yes Yes emm1 2017 Yes Yes emm28 5003 Yes Yes emm77 5004 Yes Yes emm28 5006 Yes Yes emm1 6013 Yes Yes emm1 3005 Yes No emm89 3012 Yes No emm77 6016 Yes No emm1 6018 Yes No emm1 6025 Yes No emm1 6026 Yes No emm4 6033 Yes No emm63 6028 No No emm4 6040 No No emm28 Isolate NSTI IVIG Treatment emm Type 2001 Yes Yes emm1 2006 Yes Yes emm1 2017 Yes Yes emm28 5003 Yes Yes emm77 5004 Yes Yes emm28 5006 Yes Yes emm1 6013 Yes Yes emm1 3005 Yes No emm89 3012 Yes No emm77 6016 Yes No emm1 6018 Yes No emm1 6025 Yes No emm1 6026 Yes No emm4 6033 Yes No emm63 6028 No No emm4 6040 No No emm28 Abbreviations: IVIG, intravenous immunoglobulin; NSTI, necrotizing soft-tissue skin infection. View Large Figure 2. View largeDownload slide Phagocytic killing of Streptococcus pyogenes isolates from patients with necrotizing soft-tissue infection (NSTI) or nonnecrotizing STI, opsonized with intravenous immunoglobulin (IVIG) or patient plasma. A, Survival of S. pyogenes isolates from NSTI and nonnecrotizing STI cases after incubation for 3 hours with human granulocytes and opsonization with the corresponding human plasma sample. Plasma specimens were collected at the time of admission to the hospital (gray bars) and 3 days after admission (black bars). Each bar represents the bacterial cell density as a percentage of the net growth in the absence of granulocytes. Data represent arithmetic means ± standard deviations of two individual experiments. B, Survival of S. pyogenes isolates from NSTI and nonnecrotizing STI cases after incubation for 3 hours with human granulocytes and in the presence (black bars) or absence (gray bars) of IVIG. Each bar represents the bacterial cell density as a percentage of the net growth in the absence of granulocytes. Data represents arithmetic means ± standard deviations of two individual experiments. *P < .05, by a 2-tailed paired Student t test, for the difference in bacterial survival in the presence and absence of human granulocytes. Figure 2. View largeDownload slide Phagocytic killing of Streptococcus pyogenes isolates from patients with necrotizing soft-tissue infection (NSTI) or nonnecrotizing STI, opsonized with intravenous immunoglobulin (IVIG) or patient plasma. A, Survival of S. pyogenes isolates from NSTI and nonnecrotizing STI cases after incubation for 3 hours with human granulocytes and opsonization with the corresponding human plasma sample. Plasma specimens were collected at the time of admission to the hospital (gray bars) and 3 days after admission (black bars). Each bar represents the bacterial cell density as a percentage of the net growth in the absence of granulocytes. Data represent arithmetic means ± standard deviations of two individual experiments. B, Survival of S. pyogenes isolates from NSTI and nonnecrotizing STI cases after incubation for 3 hours with human granulocytes and in the presence (black bars) or absence (gray bars) of IVIG. Each bar represents the bacterial cell density as a percentage of the net growth in the absence of granulocytes. Data represents arithmetic means ± standard deviations of two individual experiments. *P < .05, by a 2-tailed paired Student t test, for the difference in bacterial survival in the presence and absence of human granulocytes. Patients With NSTIs Lack Specific Antibodies Against the Corresponding S. pyogenes Exotoxins Since streptococcal exotoxins have been reported to play an important role in the development and severity of NSTI [14], the 16 S. pyogenes isolates were analyzed for the presence of genes coding for 15 streptococcal exotoxins. Of the 11 genes reported to encode factors with superantigenic properties (Figure 3A), only speG was present in all isolates. Also, speB and ska could be detected in the genomes of all isolates, independent of whether they originated from NSTI cases or nonnecrotizing STI cases (Figure 3A). However, there was no specific streptococcal exotoxin profile associated with the development of NSTI. Figure 3. View largeDownload slide Antibodies titers against Streptococcus pyogenes exotoxins in patient plasma and intravenous immunoglobulin (IVIG) preparations. A, Exotoxin genes present in S. pyogenes strains isolated from patients with necrotizing soft-tissue infections (NSTIs) and those with nonnecrotizing STIs. The presence (+) or absence (blank) of 17 genes coding for streptococcal exotoxins was determined in the genomes of S. pyogenes strains isolated from selected patients with NSTI or nonnecrotizing STI. In case of the ska gene, the sequence type is indicated. The similarity in the exotoxin gene profile between strains was calculated using the Bray Curtis algorithm, and the depicted dendrogram was constructed by agglomerative hierarchical clustering (group average). B, Antibody titers against streptococcal exotoxins in patient plasma. Antibody titers were determined against 18 streptococcal exotoxins identified in the corresponding S. pyogenes isolates from patients with NSTI and those with nonnecrotizing STI, using patient plasma samples collected on admission to the hospital (left) and 3 days after admission (right). Signal intensities were recorded at an absorbance of 416 nm against a negative control consisting of an empty vector used for protein purification, to exclude the background signal produced by potential contamination from the Escherichia coli M15 expression system. The difference in antibody titers between patient plasma samples and the corresponding background control was analyzed by analysis of variance (ANOVA; P < .05). Red, exotoxin gene is present in the bacterial genome but no significant antibody titer is in the corresponding patient plasma sample; blue, gene is present in the bacterial genome and an antibody titer is present in plasma; gray, gene is absent in the bacterial genome but antibody titers are detectable in plasma; white, gene is absent in the bacterial genome and antibody titers are undetectable in plasma. C, Antibody titers against streptococcal exotoxins in IVIG preparations. The antibody titer in IVIG preparations against 18 streptococcal exotoxins was determined by enzyme-linked immunosorbent analysis (gray bars). The background signal was determined using an empty vector protein purification system (black bars). Each bar represents the arithmetic mean ± standard deviation. The difference in the determined antibody titer in IVIG preparations between the streptococcal toxin and the corresponding background control was analyzed by ANOVA (*P < .05). PCR, polymerase chain reaction. Figure 3. View largeDownload slide Antibodies titers against Streptococcus pyogenes exotoxins in patient plasma and intravenous immunoglobulin (IVIG) preparations. A, Exotoxin genes present in S. pyogenes strains isolated from patients with necrotizing soft-tissue infections (NSTIs) and those with nonnecrotizing STIs. The presence (+) or absence (blank) of 17 genes coding for streptococcal exotoxins was determined in the genomes of S. pyogenes strains isolated from selected patients with NSTI or nonnecrotizing STI. In case of the ska gene, the sequence type is indicated. The similarity in the exotoxin gene profile between strains was calculated using the Bray Curtis algorithm, and the depicted dendrogram was constructed by agglomerative hierarchical clustering (group average). B, Antibody titers against streptococcal exotoxins in patient plasma. Antibody titers were determined against 18 streptococcal exotoxins identified in the corresponding S. pyogenes isolates from patients with NSTI and those with nonnecrotizing STI, using patient plasma samples collected on admission to the hospital (left) and 3 days after admission (right). Signal intensities were recorded at an absorbance of 416 nm against a negative control consisting of an empty vector used for protein purification, to exclude the background signal produced by potential contamination from the Escherichia coli M15 expression system. The difference in antibody titers between patient plasma samples and the corresponding background control was analyzed by analysis of variance (ANOVA; P < .05). Red, exotoxin gene is present in the bacterial genome but no significant antibody titer is in the corresponding patient plasma sample; blue, gene is present in the bacterial genome and an antibody titer is present in plasma; gray, gene is absent in the bacterial genome but antibody titers are detectable in plasma; white, gene is absent in the bacterial genome and antibody titers are undetectable in plasma. C, Antibody titers against streptococcal exotoxins in IVIG preparations. The antibody titer in IVIG preparations against 18 streptococcal exotoxins was determined by enzyme-linked immunosorbent analysis (gray bars). The background signal was determined using an empty vector protein purification system (black bars). Each bar represents the arithmetic mean ± standard deviation. The difference in the determined antibody titer in IVIG preparations between the streptococcal toxin and the corresponding background control was analyzed by ANOVA (*P < .05). PCR, polymerase chain reaction. To analyze whether the levels of antibodies against the corresponding streptococcal exotoxins in plasma samples may differ between patients with IVIG-treated NSTIs, those with non–IVIG-treated NSTIs, and those with nonnecrotizing STIs (Supplementary Data Set 1), an ELISA-based IgG detection system was established using 14 streptococcal exotoxins cloned, overexpressed, and purified in this study, as well as the commercially available cysteine protease SpeB, SLO, and Hyl (Supplementary Figure 1). Only minor differences in the levels of detectable IgG titers against streptococcal exotoxins were observed in day 0 plasma samples from IVIG-treated or nontreated patients with NSTIs (Figure 3B). IgG titers were detectable against only a fraction of the total number of toxins encoded in the genome of the infecting S. pyogenes strain for IVIG-treated and nontreated NSTI cases (Figure 3B). In contrast, IgG titers against all exotoxins encoded by the respective infecting S. pyogenes strain were observed in day 0 plasma specimens collected from both patients in whom the initial STI did not develop into a NSTI (Figure 3B). The antibody profile in plasma samples collected on day 3 of infection indicated that IVIG-untreated patients had increased antibody titers only against some of the toxins encoded in the genomes of the infecting S. pyogenes strains and that IgG titers were still absent against a proportion of exotoxins, including the superantigens SpeI, SpeJ, SpeK, SpeL, and SpeM (Figure 3B). In contrast, the day 3 plasma samples collected from patients with NSTIs who received IVIG treatment displayed detectable antibodies titers against all toxins except SpeG (Figure 3B). These results indicate that IVIG treatment provides patients with NSTIs with a pool of antibodies against a wide array of exotoxins that complement those that may not be induced by natural infection. Indeed, IVIG preparations contained detectable antibody titers against all exotoxins tested in this study (Figure 3C). The antibody profile of plasma samples from patients with nonnecrotizing STIs showed no changes during the course of infection, since all streptococcal exotoxins encoded in the genomes of the infecting S. pyogenes strains were covered by specific antibodies already in day 0 plasma samples (Figure 3B). This may suggest that preexisting antibodies against the repertoire of exotoxins produced by the infecting S. pyogenes strain may prevent the progression of STI to severe NSTI. The opposite could also be the case, and patients lacking antibodies against specific S. pyogenes exotoxins may be at risk for developing NSTI. Patients With NSTIs Lack Antibodies That Neutralize the Effect of S. pyogenes Exotoxins Many of the devastating effects of S. pyogenes NSTI are mediated by the hyperactivation of T cells by streptococcal superantigens, as well as by the tissue damage caused by toxins such as the streptococcal cytolysin SLO, SpeB, and Sic [15]. To determine whether the antibodies present in the plasma of patients with NSTIs are capable to neutralize the effect of these factors, functional assays were performed using plasma samples collected from a representative patient subset. Plasma samples that contained antibodies against specific streptococcal superantigens were highly effective at inhibiting the superantigenicity effect on T-cell proliferation (>90%) of the specific superantigen (Figure 4A and Supplementary Data Set 2). Only in the case of SpeA the inhibition was incomplete (42%–84%). Among plasma lacking antibodies against the specific superantigen, the inhibition never exceeded 12% (Figure 4A). Similar to superantigens, the capacity of a patient’s plasma specimen to suppress the functional effect of SpeB, SLO, and Sic strongly correlated with the presence of antibodies specific for the corresponding toxin (Figure 4A). This indicates that patients developing NSTI did not carry specific antibodies against many of the exotoxins potentially secreted by the infecting bacterial isolate and thus could not inhibit the negative effect of these toxins. In contrast, the antibodies detected in day 0 samples from patients with nonnecrotizing STIs were capable of inhibiting exotoxins encoded by the invading pathogen, and neither change in antibody titers nor in inhibition potential was observed during infection progression (Figure 4A). Notably, plasma specimens from patients who received IVIG treatment were capable of inhibiting the activity of all analyzed exotoxins, with the exception of that of the superantigen SpeA (Figure 4A). To evaluate whether this effect was mediated by toxin-neutralizing antibodies contained in the IVIG preparations, functional assays were performed with IgG-depleted human plasma supplemented with either 1 mg/mL or 5 mg/mL of IVIG. The induction of T-cell proliferation by streptococcal superantigens (SpeA, SpeC, SpeG, SpeH, SpeI, SpeJ, SpeK, SpeL, SpeM, SSA, and SmeZ) was clearly inhibited (>90%) by IVIG even at a concentration of only 20% of the dosage used in clinical settings (1 mg/mL; Figure 4B). SpeA was the only superantigen whose activity was not fully inhibited; however, 73% inhibition was observed when IVIG was administered at the concentration used in the clinic (5 mg/mL; Figure 4B). The activity of the streptococcal toxins SpeB, SLO, and Sic was also effectively neutralized by the clinically relevant concentration of IVIG (Figure 4B). This demonstrates that IVIG preparations contain antibodies against 14 streptococcal exotoxins that, when used in the clinically appropriate concentration, are able to completely inhibit the function of 13 of them, leaving only SpeA as partially inhibited. Figure 4. View largeDownload slide Exotoxin neutralizing capacity of plasma and intravenous immunoglobulin (IVIG) preparations. A, Antibody titers against streptococcal exotoxins in patient plasma and their capability to inhibit the exotoxins function. Antibody titers were determined against 14 streptococcal exotoxins identified in the corresponding Streptococcus pyogenes isolates from patients with necrotizing soft-tissue infection (NSTI) or nonnecrotizing STI, using plasma samples collected upon admission to the hospital (day 0) and 3 days after admission (day 3). Toxin activities were determined by a T-cell proliferation assay (for superantigens), an LL-37 protection assay (for Sic), an azocasein assay (for SpeB), or an erythrocyte cytotoxicity assay (for streptolysin O [SLO]) after incubation in the corresponding patient plasma sample. Percentage inhibition was calculated relative to the 100% toxin activity determined in immunoglobulin G (IgG)–depleted plasma and is indicated in the respective boxes. Red, exotoxin gene is present in the bacterial genome but no significant antibody titer is in the corresponding patient plasma sample; blue, gene is present in the bacterial genome and an antibody titer is present in plasma; gray, gene is absent in the bacterial genome but antibody titers are detectable in plasma; white, gene is absent in the bacterial genome and antibody titers are undetectable in plasma. B, Inhibitory capacity of IVIG preparations of 14 streptococcal exotoxins. Toxin activities were determined by a T-cell proliferation assay (for superantigens), an azocasein assay (for SpeB), an LL-37 protection assay (for Sic), or an erythrocyte cytotoxicity assay (for SLO) after incubation in IgG-depleted human plasma containing 1 mg/mL IVIG (gray bars) or 5 mg/mL IVIG (black bars). Inhibition is calculated relative to the 100% activity determined in IgG-depleted plasma without IVIG. Data represent arithmetic means ± standard deviations of two individual experiments. Figure 4. View largeDownload slide Exotoxin neutralizing capacity of plasma and intravenous immunoglobulin (IVIG) preparations. A, Antibody titers against streptococcal exotoxins in patient plasma and their capability to inhibit the exotoxins function. Antibody titers were determined against 14 streptococcal exotoxins identified in the corresponding Streptococcus pyogenes isolates from patients with necrotizing soft-tissue infection (NSTI) or nonnecrotizing STI, using plasma samples collected upon admission to the hospital (day 0) and 3 days after admission (day 3). Toxin activities were determined by a T-cell proliferation assay (for superantigens), an LL-37 protection assay (for Sic), an azocasein assay (for SpeB), or an erythrocyte cytotoxicity assay (for streptolysin O [SLO]) after incubation in the corresponding patient plasma sample. Percentage inhibition was calculated relative to the 100% toxin activity determined in immunoglobulin G (IgG)–depleted plasma and is indicated in the respective boxes. Red, exotoxin gene is present in the bacterial genome but no significant antibody titer is in the corresponding patient plasma sample; blue, gene is present in the bacterial genome and an antibody titer is present in plasma; gray, gene is absent in the bacterial genome but antibody titers are detectable in plasma; white, gene is absent in the bacterial genome and antibody titers are undetectable in plasma. B, Inhibitory capacity of IVIG preparations of 14 streptococcal exotoxins. Toxin activities were determined by a T-cell proliferation assay (for superantigens), an azocasein assay (for SpeB), an LL-37 protection assay (for Sic), or an erythrocyte cytotoxicity assay (for SLO) after incubation in IgG-depleted human plasma containing 1 mg/mL IVIG (gray bars) or 5 mg/mL IVIG (black bars). Inhibition is calculated relative to the 100% activity determined in IgG-depleted plasma without IVIG. Data represent arithmetic means ± standard deviations of two individual experiments. DISCUSSION The development of a NSTI is characterized by a complex interplay between the host immune system and the invading pathogen that produces a wide array of virulence factors to evade and manipulate the immune response. Owing to the crucial role of antibodies in host defense against S. pyogenes [9], the present study investigated the role of pathogen-specific antibodies in the development and progression of S. pyogenes NSTI. Although the microbiologic background of NSTI is considerably diverse [1], we confirmed the previously reported dominating role of S. pyogenes as an etiologic agent, especially those of emm type emm1 [1, 16–19]. The clinical isolates in this study also included emm types more rarely observed in invasive infections such as emm77 and emm63 [18, 20]. Although S. pyogenes is a frequent cause of skin infections, the reason why in particular patients, these infections progress into severe NSTI remains unclear. The results of this study indicate that the absence of preexisting antibody titers against pathogen-specific determinants in patient sera during the early stage of tissue infection may represent a risk factor for the development of a NSTI. This was substantiated by the observation that opsonophagocytic killing rates of the infecting S. pyogenes strain by the corresponding patient plasma specimen collected on day 0 were significantly lower in patients with NSTIs than in nonnecrotizing STI control cases. These observations suggest a direct link between an individual’s serologic profile and their risk of progression into severe NSTI. We also provide evidence that deficiency of pathogen-specific opsonic antibodies in patients with NSTIs can be compensated, at least to some extent, by the administration of IVIG. Streptococcal NSTI is frequently treated with IVIG, a pool of concentrated human serum antibodies [19]. Even though few reports suggested that IVIG contains protective antibody titers against S. pyogenes, its efficacy in the NSTI scenario remains ambiguous [21]. As many of the adverse effects associated with the progression of NSTI are mediated by the activity of superantigen and toxins [22–24], the presence of antibodies capable of neutralizing the activity of these bacterial factors was also investigated here. Interestingly, no difference in the exotoxin gene profile between nonnecrotizing STI and NSTI bacterial isolates was observed, confirming that differences in virulence factor profiles may explain the invasiveness and outcome of S. pyogenes infections only to a limited extent [25]. IVIG have been so far only investigated for the presence of antibodies against the superantigens SpeA, SpeC, and SpeG [26]. We could identify significant antibody titers against 14 additional toxins potentially involved in the development of NSTI. The combined effect of enhancing opsonophagocytic killing and neutralizing the full array of bacterial superantigens and toxins highlights the potential benefit of using IVIG as adjunctive therapy for limiting the progression of NSTI. However, IVIG therapy seems to be of benefit only if administered during the very early stage of NSTI, since administration of IVIG at later stages of infection will not invert the progression of an already established NSTI. This may explain the results obtained from clinical studies, where no significant effect of IVIG treatment on the outcome and mortality of NSTI was observed [21, 27]. The antibody profile observed in the non–IVIG-treated NSTI cases clearly discriminates between bacterial factors able to induce a robust antigen-specific antibody response, like SpeA, SpeC, SpeG, SpeH, or SmeZ, and nonimmunogenic toxins, such as SpeI, SpeJ, SpeK, SpeL, and SpeM, which failed to induce an antibody response. These differences in antibody levels during the acute phase of infection probably reflect a preexisting memory B- and T-cell response only against a set of bacterial antigens that can be activated very fast after a second exposure to the pathogen [28]. We can, therefore, hypothesize that the subset of analyzed bacterial toxins, which does not induce a specific antibody response, is either not expressed during the infection process and thereby not involved in the NSTI progression or that the patient has never been confronted with a pathogen producing these factors and therefore lacks specific memory responses. Although S. pyogenes strains carrying speL, speM, and speK have been more regularly isolated from cases of acute rheumatic fever [29, 30], a direct association of these superantigens with invasive streptococcal infections like NSTI is not known and should be addressed in future studies. Antibodies can mediate protection against streptococcal toxins by neutralizing the activity of a specific toxin. Using functional assays, we demonstrated the efficiency of IVIG, used at a clinically relevant dosage, in neutralizing 13 streptococcal exotoxins. The observation that the potent superantigen SpeA cannot be blocked completely by antibodies is in accordance with previous studies [31] and is discussed to be linked to its binding specificity to the major histocompatibility complex class II receptor α chain, which discriminates SpeA from all other streptococcal superantigens [32]. In summary, the results presented here are of major clinical relevance. Even though the incidence of NSTI is low, increasing numbers of affected cases have been observed [33]. The dramatic manifestation, unclear origin, and complicated diagnosis of this life-threatening infection make the identification of risk factors a major challenge. The study presented here identified a state of serologic susceptibility potentially linked to the irreversible progression of an initial tissue infection to a severe NSTI. This interpretation suggests that a quick intervention with a high dose of IVIG during the early stage is potentially able to interfere with infection progression and, in that way, not only positively influences the efficiency of other antimicrobial treatment strategies but also decreases the risk of side effects, thereby increasing the chance of survival among affected patients. STUDY GROUP MEMBERS Members of the INFECT study group are as follows: Anna Norrby-Teglund, Mattias Svensson, Anders Rosén, Ylva Karlsson, Martin B Madsen, Steinar Skrede, Oddvar Oppegaard, Torbjørn Nedrebø, and Eva Medina. Affiliations for all study group members are specified in the Supplementary Materials. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Acknowledgments.  We thank the hospital teams in Denmark, Sweden, and Norway that were involved in INFECT project, for their help with patient inclusion, collection of blood specimens, and entering of clinical information into the database; Dr Giuseppe Gulotta, for providing the modified pQE30 vector containing the TEV cleavage site (pQE30-TEV); Dr René Bergmann, for his technical help and fruitful discussions; and Dr Eva Medina and Dr Oliver Goldmann, for their critical review of manuscript and scientific input. Author contributions. A. I. designed the study. A. B. and A. I. performed the experiments and analyzed the data. The INFECT Study Group contributed to clinical study design, study planning, patient recruitment, patient sample collection, typing, and patient data analyses. A. B., A. I., and D. H. P. wrote the manuscript. All authors read and approved the final version. Financial support. This work was supported by the European Union’s Seventh Framework Program (grant 305340). Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Presented in part: 20th Lancefield International Symposium on Streptococci and Streptococcal Disease, 16–20 October 2017, Fiji. References 1. Misiakos EP , Bagias G , Patapis P , Sotiropoulos D , Kanavidis P , Machairas A . Current concepts in the management of necrotizing fasciitis . Front Surg 2014 ; 1 – 36 . 2. Bisno AL , Stevens DL . Streptococcal infections of skin and soft tissues . N Engl J Med 1996 ; 334 : 240 – 5 . Google Scholar CrossRef Search ADS PubMed 3. Wyrick W Jr . Necrotizing fasciitis . J Am Acad Dermatol 1986 ; 15 : 299 – 300 . Google Scholar CrossRef Search ADS PubMed 4 . Barnham MR , Weightman NC , Anderson AW , Tanna A . Streptococcal toxic shock syndrome: a description of 14 cases from North Yorkshire, UK . Clin Microbiol Infect 2002 ; 8 : 174 – 81 . Google Scholar CrossRef Search ADS PubMed 5. Kaul R , McGeer A , Low DE , Green K , Schwartz B . Population-based surveillance for group A streptococcal necrotizing fasciitis: clinical features, prognostic indicators, and microbiologic analysis of seventy-seven cases. Ontario Group A Streptococcal Study . Am J Med 1997 ; 103 : 18 – 24 . Google Scholar CrossRef Search ADS PubMed 6. Bruun T , Kittang BR , de Hoog BJ et al. Necrotizing soft tissue infections caused by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis of groups C and G in western Norway . Clin Microbiol Infect 2013 ; 19 : E545 – 50 . Google Scholar CrossRef Search ADS PubMed 7. Olsen RJ , Musser JM . Molecular pathogenesis of necrotizing fasciitis . Annu Rev Pathol 2010 ; 5 : 1 – 31 . Google Scholar CrossRef Search ADS PubMed 8. Huang KF , Hung MH , Lin YS et al. Independent predictors of mortality for necrotizing fasciitis: a retrospective analysis in a single institution . J Trauma 2011 ; 71 : 467 – 73 . Google Scholar CrossRef Search ADS PubMed 9. Basma H , Norrby-Teglund A , McGeer A et al. Opsonic antibodies to the surface M protein of group A streptococci in pooled normal immunoglobulins (IVIG): potential impact on the clinical efficacy of IVIG therapy for severe invasive group A streptococcal infections . Infect Immun 1998 ; 66 : 2279 – 83 . Google Scholar PubMed 10. Babbar A , Kumar VN , Bergmann R et al. Members of a new subgroup of Streptococcus anginosus harbor virulence related genes previously observed in Streptococcus pyogenes . Int J Med Microbiol 2017 ; 307 : 174 – 81 . Google Scholar CrossRef Search ADS PubMed 11. Collin M , Olsén A . Generation of a mature streptococcal cysteine proteinase is dependent on cell wall-anchored M1 protein . Mol Microbiol 2000 ; 36 : 1306 – 18 . Google Scholar CrossRef Search ADS PubMed 12. Ruiz N , Wang B , Pentland A , Caparon M . Streptolysin O and adherence synergistically modulate proinflammatory responses of keratinocytes to group A streptococci . Mol Microbiol 1998 ; 27 : 337 – 46 . Google Scholar CrossRef Search ADS PubMed 13. Frick IM , Akesson P , Rasmussen M , Schmidtchen A , Björck L . SIC, a secreted protein of Streptococcus pyogenes that inactivates antibacterial peptides . J Biol Chem 2003 ; 278 : 16561 – 6 . Google Scholar CrossRef Search ADS PubMed 14. Babbar A. Streptococcal superantigens . 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Intensive Care Med 2017 ; 43 : 1585 – 93 . Google Scholar CrossRef Search ADS PubMed 22. Walker MJ , Barnett TC , McArthur JD et al. Disease manifestations and pathogenic mechanisms of group A Streptococcus . Clin Microbiol Rev 2014 ; 27 : 264 – 301 . Google Scholar CrossRef Search ADS PubMed 23. Borek AL , Obszańska K , Hryniewicz W , Sitkiewicz I . Detection of Streptococcus pyogenes virulence factors by multiplex PCR . Virulence 2012 ; 3 : 529 – 33 . Google Scholar CrossRef Search ADS PubMed 24. Borek AL , Obszańska K , Hryniewicz W , Sitkiewicz I . Typing of Streptococcus pyogenes strains using the phage profiling method . Virulence 2012 ; 3 : 534 – 8 . Google Scholar CrossRef Search ADS PubMed 25. Chatellier S , Ihendyane N , Kansal RG et al. Genetic relatedness and superantigen expression in group A streptococcus serotype M1 isolates from patients with severe and nonsevere invasive diseases . Infect Immun 2000 ; 68 : 3523 – 34 . Google Scholar CrossRef Search ADS PubMed 26. Norrby-Teglund A , Kaul R , Low DE et al. Evidence for the presence of streptococcal-superantigen-neutralizing antibodies in normal polyspecific immunoglobulin G . Infect Immun 1996 ; 64 : 5395 – 8 . Google Scholar PubMed 27. Kadri SS , Swihart BJ , Bonne SL et al. Impact of intravenous immunoglobulin on survival in necrotizing fasciitis with vasopressor-dependent shock: a propensity score-matched analysis from 130 US hospitals . Clin Infect Dis 2017 ; 64 : 877 – 85 . Google Scholar PubMed 28. Kuby J , Goldsby R , Kindt T , Osborne B. Immunology . 5th ed . 2002 . New York : W.H. Freeman and Company . 29. Smoot JC , Barbian KD , Van Gompel JJ et al. Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks . Proc Natl Acad Sci U S A 2002 ; 99 : 4668 – 73 . Google Scholar CrossRef Search ADS PubMed 30. Proft T , Sriskandan S , Yang L , Fraser JD . Superantigens and streptococcal toxic shock syndrome . Emerg Infect Dis 2003 ; 9 : 1211 – 8 . Google Scholar CrossRef Search ADS PubMed 31. Norrby-Teglund A , Basma H , Andersson J , McGeer A , Low DE , Kotb M . Varying titers of neutralizing antibodies to streptococcal superantigens in different preparations of normal polyspecific immunoglobulin G: implications for therapeutic efficacy . Clin Infect Dis 1998 ; 26 : 631 – 8 . Google Scholar CrossRef Search ADS PubMed 32. Proft T , Fraser JD . Bacterial superantigens . Clin Exp Immunol 2003 ; 133 : 299 – 306 . Google Scholar CrossRef Search ADS PubMed 33. Wang JM , Lim HK . Necrotizing fasciitis: eight-year experience and literature review . Braz J Infect Dis 2014 ; 18 : 137 – 43 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Pivotal Role of Preexisting Pathogen-Specific Antibodies in the Development of Necrotizing Soft-Tissue Infections

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0022-1899
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10.1093/infdis/jiy110
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Abstract

Abstract Background Necrotizing soft-tissue infections (NSTI) are the most severe form of bacterial-induced tissue pathology. Their unpredictable onset and rapid development into life-threatening conditions considerably complicate patient treatment. Understanding the risk factors for NSTI in individual patients is necessary for selecting the appropriate therapeutic option. Methods We investigated the role of pathogen-specific antibodies in the manifestation of NSTI by performing a comparative serologic approach, using plasma samples and bacterial isolates from patients with clinical NSTIs or nonnecrotizing STIs caused by Streptococcus pyogenes. We also evaluated the potential beneficial effect of intravenous immunoglobulin (IVIG) treatment. Results We identified a hitherto overlooked state of serologic susceptibility in patients with NSTIs during the earliest stages of the infection that is potentially linked to disease progression. Thus, all patients with NSTIs included in this study exhibited a deficiency in specific antibodies directed against the causative S. pyogenes strains and the majority of their exotoxins during the initial stage of the infection. We also showed that the clinical use of IVIG during the course of infection compensates the observed antibody deficiency but is unable to halt the disease progression, once tissue necrosis has developed. Conclusion These observations emphasize the requirement of preexisting pathogen-specific antibodies to prevent the irreversible progression of tissue infections into severely spreading NSTIs and urge further investigations on the beneficial effect of IVIG-based early phase intervention strategies to prevent the severe effects of this devastating bacterial infection. Antibody titers, exotoxins, intravenous immunoglobulins (IVIG), necrotizing Soft tissue infections, Streptococcus pyogenes Necrotizing soft-tissue infections (NSTIs) are the most dramatic form of bacterial-induced tissue pathology that is associated with devastating side effects, ranging from severe soft-tissue necrosis to systemic inflammation and toxic shock [1–4]. Streptococcus pyogenes is a major cause of NSTIs [5, 6]. Owing to the rapidly progressing nature of NSTI, prompt diagnosis and treatment, including aggressive surgery and antibiotics, along with possibly adjunctive therapies, such as intravenous immunoglobulin (IVIG), are critical. Limited knowledge on the abrupt onset of NSTI and on the mechanisms driving a self-limiting local tissue infection into a severely spreading necrotic stage remain a major challenge for establishing tailored strategies that interfere with NSTI development in patients at risk. Soft-tissue infections are generally initiated after a pathogen enters the hypodermal tissue through a wound or by the hematogenic spread into tissue damaged by minor trauma [7, 8]. However, the comparatively low number of tissue infections that develop into NSTI implies that the immune system is, in most instances, able to control the invading pathogen, thereby preventing the progression to NSTI. Based on these premises and on the important role of opsonic and toxin-neutralizing antibodies for controlling S. pyogenes infections [9], it can be hypothesized that serologic susceptibility characterized by a lack or low levels of specific antibodies against the pathogen and its toxins may predispose an individual to NSTI. The current study investigates the role of specific antibodies against S. pyogenes and its toxins in the development of NSTI and characterizes the impact of IVIG as adjunctive therapy in the progression of NSTI caused by S. pyogenes. This study is the first to demonstrate the protective effect of preexisting antibody titers against streptococcal surface and secreted exotoxins in preventing the irreversible progression of mild tissue infections into severe NSTI and the potential benefit of IVIG for the treatment of patients at risk. MATERIAL AND METHODS Patient Plasma Samples Patients with clinical NSTIs or nonnecrotizing STIs were recruited, and the corresponding bacterial isolates were collected within the framework of the European Union–funded project INFECT (available at: http://www.fp7infect.eu; Supplementary Table 1). Plasma samples from patients were collected when the patient was recruited in the study (day 0) and after 3 days of treatment (day 3). Written informed consent was obtained from all patients or their next of kin as soon as possible. The study is part of INFECT and is registered at ClinicalTrials.gov (NCT01790698). All samples were collected in accordance with the Declaration of Helsinki and with the approval of the regional Ethical Review Board at the Karolinska Institutet in Stockholm, Sweden (ethics permits: 2012/2110-31/2); the National Committee on Health Research Ethics in Copenhagen, Denmark (Ethics permits: 1151739); or the regional Ethical Review Board in Gothenburg, Sweden (ethics permits: 930-12) or Bergen, Norway (2012/2227/REC West). Fresh human blood samples were collected in house from the right median cubital vein of healthy middle-aged volunteers under ethical approval number BO/07/2013. Bacteria Streptococcal isolates were cultivated in Todd Hewitt broth containing 0.5% yeast extract at 37°C. Exponentially growing cultures were used (absorbance at 600 nm = 0.4) unless stated otherwise. Escherichia coli strains were cultivated in Luria-Bertani medium supplemented with appropriate concentrations of antibiotics (Supplementary Table 2) at 37°C (or 30°C after IPTG induction) horizontally shaking at 150 rpm. Clinical isolates were identified by 16S ribosomal DNA sequencing and typed for their hemolytic behavior, surface antigen presentation, and emm gene. Genomic DNA was extracted from overnight cultures and screened for the presence of 14 streptococcal exotoxin encoding genes (speA, speC, speG, speH, speI, speJ, speK, speL, speM, ssa, smeZ, speB, sic, and mac) by multiplex polymerase chain reaction (PCR) analysis as described previously [10]. All PCR products were sequenced using terminator cycle sequencing (ABI Prism). Opsonophagocytosis Assay In vitro opsonophagocytosis assays were performed using freshly isolated human granulocytes and plasma samples as previously described (Supplementary Methods). Antibody Detection and Inhibition Assays Antigen-specific immunoglobulin G (IgG) titers in patient plasma samples were determined using standard enzyme-linked immunosorbent assay (ELISA) protocols after cloning and purification of 14 streptococcal exotoxins (Supplementary Methods). SpeB (Genovis), Mac (Genovis), streptolysin O (SLO; Sigma), and hyaluronidase (Hyl; Sigma) were purchased from commercial suppliers. To characterize the neutralization of toxin effects by antibodies in IVIG or patient plasma, inhibition assays were performed. The activity of superantigens was measured using T-cell proliferation assays (Supplementary Methods). The activities of SpeB, SLO, and Sic were measured through cleavage of azocasein, red blood cell lysis, and LL-37 inhibition as previously described [11–13]. Nucleotide Sequence Accession Numbers The 16S ribosomal DNA and emm gene sequences reported in this study have been deposited in GenBank under accession numbers MF480476-MF480523 and MF538589-MF538623. Statistical Analysis Comparisons between groups were made using Student t test or by analysis of variance. P values of < .05 were considered significant. RESULTS Patients With NSTIs Lack Antibodies Against the Corresponding S. pyogenes Isolate and Its Exotoxins A cohort of 51 NSTI cases recruited within the frame of the European Union–funded project INFECT and identified as monoinfections caused by members of the genus Streptococcus were included in this study (Figure 1A). S. pyogenes was identified as the most frequent causative pathogen (63% of cases; Figure 1A). The S. pyogenes isolates comprised 11 serotypes, with emm1 being the most abundant (47%; Figure 1B). Figure 1. View largeDownload slide Species and serotype distribution of isolates from 51 necrotizing soft-tissue monoinfections caused by members of the genus Streptococcus. A, Streptococcal species distribution identified by 16S ribosomal RNA gene sequencing. B, Serotypes distribution of all 32 S. pyogenes isolates, determined by emm sequencing. Figure 1. View largeDownload slide Species and serotype distribution of isolates from 51 necrotizing soft-tissue monoinfections caused by members of the genus Streptococcus. A, Streptococcal species distribution identified by 16S ribosomal RNA gene sequencing. B, Serotypes distribution of all 32 S. pyogenes isolates, determined by emm sequencing. To investigate a potential correlation between the immunologic status of the patients and the development of NSTI, a comprehensive serologic study involving the characterization of the opsonophagocytic capacity of plasma collected at different times of infection against the corresponding bacterial isolate was performed in a representative set of 7 IVIG-treated and 7 non–IVIG-treated NSTI cases reflecting the serotype distribution of the complete set of S. pyogenes isolates. Two cases of nonnecrotizing STIs were included as study controls (Table 1). Whereas plasma samples collected on the day of study recruitment (day 0) from all 14 NSTI cases were inefficient at inducing opsonophagocytic killing of the corresponding S. pyogenes strain, plasma specimens from the 2 patients with nonnecrotizing STIs exhibited a remarkable capacity to induce opsonophagocytic elimination of S. pyogenes (Figure 2A). These findings suggest that the lack of opsonizing antibodies in patients with NSTIs may be a decisive factor for the progression from STI to NSTI. Interestingly, plasma samples collected on day 3 from all patients with NSTIs exhibited a significant increase in opsonophagocytosis activity that was more pronounced in the IVIG-treated group (Figure 2A). These observations demonstrated that opsonic antibodies are elicited in all patients with the progression of S. pyogenes infection and that IVIG treatment enhanced opsonophagocytosis of S. pyogenes, an effect that was most probably mediated by the presence of high levels of opsonic antibodies in the IVIG preparations. The presence of opsonic antibodies in IVIG preparations was demonstrated by the significant increase in bacterial killing observed after incubating the different S. pyogenes isolates with neutrophils in the presence of IVIG preparations (Figure 2B). Table 1. Cases of Streptococcus pyogenes Monoinfection Selected for the Serologic Study Isolate NSTI IVIG Treatment emm Type 2001 Yes Yes emm1 2006 Yes Yes emm1 2017 Yes Yes emm28 5003 Yes Yes emm77 5004 Yes Yes emm28 5006 Yes Yes emm1 6013 Yes Yes emm1 3005 Yes No emm89 3012 Yes No emm77 6016 Yes No emm1 6018 Yes No emm1 6025 Yes No emm1 6026 Yes No emm4 6033 Yes No emm63 6028 No No emm4 6040 No No emm28 Isolate NSTI IVIG Treatment emm Type 2001 Yes Yes emm1 2006 Yes Yes emm1 2017 Yes Yes emm28 5003 Yes Yes emm77 5004 Yes Yes emm28 5006 Yes Yes emm1 6013 Yes Yes emm1 3005 Yes No emm89 3012 Yes No emm77 6016 Yes No emm1 6018 Yes No emm1 6025 Yes No emm1 6026 Yes No emm4 6033 Yes No emm63 6028 No No emm4 6040 No No emm28 Abbreviations: IVIG, intravenous immunoglobulin; NSTI, necrotizing soft-tissue skin infection. View Large Table 1. Cases of Streptococcus pyogenes Monoinfection Selected for the Serologic Study Isolate NSTI IVIG Treatment emm Type 2001 Yes Yes emm1 2006 Yes Yes emm1 2017 Yes Yes emm28 5003 Yes Yes emm77 5004 Yes Yes emm28 5006 Yes Yes emm1 6013 Yes Yes emm1 3005 Yes No emm89 3012 Yes No emm77 6016 Yes No emm1 6018 Yes No emm1 6025 Yes No emm1 6026 Yes No emm4 6033 Yes No emm63 6028 No No emm4 6040 No No emm28 Isolate NSTI IVIG Treatment emm Type 2001 Yes Yes emm1 2006 Yes Yes emm1 2017 Yes Yes emm28 5003 Yes Yes emm77 5004 Yes Yes emm28 5006 Yes Yes emm1 6013 Yes Yes emm1 3005 Yes No emm89 3012 Yes No emm77 6016 Yes No emm1 6018 Yes No emm1 6025 Yes No emm1 6026 Yes No emm4 6033 Yes No emm63 6028 No No emm4 6040 No No emm28 Abbreviations: IVIG, intravenous immunoglobulin; NSTI, necrotizing soft-tissue skin infection. View Large Figure 2. View largeDownload slide Phagocytic killing of Streptococcus pyogenes isolates from patients with necrotizing soft-tissue infection (NSTI) or nonnecrotizing STI, opsonized with intravenous immunoglobulin (IVIG) or patient plasma. A, Survival of S. pyogenes isolates from NSTI and nonnecrotizing STI cases after incubation for 3 hours with human granulocytes and opsonization with the corresponding human plasma sample. Plasma specimens were collected at the time of admission to the hospital (gray bars) and 3 days after admission (black bars). Each bar represents the bacterial cell density as a percentage of the net growth in the absence of granulocytes. Data represent arithmetic means ± standard deviations of two individual experiments. B, Survival of S. pyogenes isolates from NSTI and nonnecrotizing STI cases after incubation for 3 hours with human granulocytes and in the presence (black bars) or absence (gray bars) of IVIG. Each bar represents the bacterial cell density as a percentage of the net growth in the absence of granulocytes. Data represents arithmetic means ± standard deviations of two individual experiments. *P < .05, by a 2-tailed paired Student t test, for the difference in bacterial survival in the presence and absence of human granulocytes. Figure 2. View largeDownload slide Phagocytic killing of Streptococcus pyogenes isolates from patients with necrotizing soft-tissue infection (NSTI) or nonnecrotizing STI, opsonized with intravenous immunoglobulin (IVIG) or patient plasma. A, Survival of S. pyogenes isolates from NSTI and nonnecrotizing STI cases after incubation for 3 hours with human granulocytes and opsonization with the corresponding human plasma sample. Plasma specimens were collected at the time of admission to the hospital (gray bars) and 3 days after admission (black bars). Each bar represents the bacterial cell density as a percentage of the net growth in the absence of granulocytes. Data represent arithmetic means ± standard deviations of two individual experiments. B, Survival of S. pyogenes isolates from NSTI and nonnecrotizing STI cases after incubation for 3 hours with human granulocytes and in the presence (black bars) or absence (gray bars) of IVIG. Each bar represents the bacterial cell density as a percentage of the net growth in the absence of granulocytes. Data represents arithmetic means ± standard deviations of two individual experiments. *P < .05, by a 2-tailed paired Student t test, for the difference in bacterial survival in the presence and absence of human granulocytes. Patients With NSTIs Lack Specific Antibodies Against the Corresponding S. pyogenes Exotoxins Since streptococcal exotoxins have been reported to play an important role in the development and severity of NSTI [14], the 16 S. pyogenes isolates were analyzed for the presence of genes coding for 15 streptococcal exotoxins. Of the 11 genes reported to encode factors with superantigenic properties (Figure 3A), only speG was present in all isolates. Also, speB and ska could be detected in the genomes of all isolates, independent of whether they originated from NSTI cases or nonnecrotizing STI cases (Figure 3A). However, there was no specific streptococcal exotoxin profile associated with the development of NSTI. Figure 3. View largeDownload slide Antibodies titers against Streptococcus pyogenes exotoxins in patient plasma and intravenous immunoglobulin (IVIG) preparations. A, Exotoxin genes present in S. pyogenes strains isolated from patients with necrotizing soft-tissue infections (NSTIs) and those with nonnecrotizing STIs. The presence (+) or absence (blank) of 17 genes coding for streptococcal exotoxins was determined in the genomes of S. pyogenes strains isolated from selected patients with NSTI or nonnecrotizing STI. In case of the ska gene, the sequence type is indicated. The similarity in the exotoxin gene profile between strains was calculated using the Bray Curtis algorithm, and the depicted dendrogram was constructed by agglomerative hierarchical clustering (group average). B, Antibody titers against streptococcal exotoxins in patient plasma. Antibody titers were determined against 18 streptococcal exotoxins identified in the corresponding S. pyogenes isolates from patients with NSTI and those with nonnecrotizing STI, using patient plasma samples collected on admission to the hospital (left) and 3 days after admission (right). Signal intensities were recorded at an absorbance of 416 nm against a negative control consisting of an empty vector used for protein purification, to exclude the background signal produced by potential contamination from the Escherichia coli M15 expression system. The difference in antibody titers between patient plasma samples and the corresponding background control was analyzed by analysis of variance (ANOVA; P < .05). Red, exotoxin gene is present in the bacterial genome but no significant antibody titer is in the corresponding patient plasma sample; blue, gene is present in the bacterial genome and an antibody titer is present in plasma; gray, gene is absent in the bacterial genome but antibody titers are detectable in plasma; white, gene is absent in the bacterial genome and antibody titers are undetectable in plasma. C, Antibody titers against streptococcal exotoxins in IVIG preparations. The antibody titer in IVIG preparations against 18 streptococcal exotoxins was determined by enzyme-linked immunosorbent analysis (gray bars). The background signal was determined using an empty vector protein purification system (black bars). Each bar represents the arithmetic mean ± standard deviation. The difference in the determined antibody titer in IVIG preparations between the streptococcal toxin and the corresponding background control was analyzed by ANOVA (*P < .05). PCR, polymerase chain reaction. Figure 3. View largeDownload slide Antibodies titers against Streptococcus pyogenes exotoxins in patient plasma and intravenous immunoglobulin (IVIG) preparations. A, Exotoxin genes present in S. pyogenes strains isolated from patients with necrotizing soft-tissue infections (NSTIs) and those with nonnecrotizing STIs. The presence (+) or absence (blank) of 17 genes coding for streptococcal exotoxins was determined in the genomes of S. pyogenes strains isolated from selected patients with NSTI or nonnecrotizing STI. In case of the ska gene, the sequence type is indicated. The similarity in the exotoxin gene profile between strains was calculated using the Bray Curtis algorithm, and the depicted dendrogram was constructed by agglomerative hierarchical clustering (group average). B, Antibody titers against streptococcal exotoxins in patient plasma. Antibody titers were determined against 18 streptococcal exotoxins identified in the corresponding S. pyogenes isolates from patients with NSTI and those with nonnecrotizing STI, using patient plasma samples collected on admission to the hospital (left) and 3 days after admission (right). Signal intensities were recorded at an absorbance of 416 nm against a negative control consisting of an empty vector used for protein purification, to exclude the background signal produced by potential contamination from the Escherichia coli M15 expression system. The difference in antibody titers between patient plasma samples and the corresponding background control was analyzed by analysis of variance (ANOVA; P < .05). Red, exotoxin gene is present in the bacterial genome but no significant antibody titer is in the corresponding patient plasma sample; blue, gene is present in the bacterial genome and an antibody titer is present in plasma; gray, gene is absent in the bacterial genome but antibody titers are detectable in plasma; white, gene is absent in the bacterial genome and antibody titers are undetectable in plasma. C, Antibody titers against streptococcal exotoxins in IVIG preparations. The antibody titer in IVIG preparations against 18 streptococcal exotoxins was determined by enzyme-linked immunosorbent analysis (gray bars). The background signal was determined using an empty vector protein purification system (black bars). Each bar represents the arithmetic mean ± standard deviation. The difference in the determined antibody titer in IVIG preparations between the streptococcal toxin and the corresponding background control was analyzed by ANOVA (*P < .05). PCR, polymerase chain reaction. To analyze whether the levels of antibodies against the corresponding streptococcal exotoxins in plasma samples may differ between patients with IVIG-treated NSTIs, those with non–IVIG-treated NSTIs, and those with nonnecrotizing STIs (Supplementary Data Set 1), an ELISA-based IgG detection system was established using 14 streptococcal exotoxins cloned, overexpressed, and purified in this study, as well as the commercially available cysteine protease SpeB, SLO, and Hyl (Supplementary Figure 1). Only minor differences in the levels of detectable IgG titers against streptococcal exotoxins were observed in day 0 plasma samples from IVIG-treated or nontreated patients with NSTIs (Figure 3B). IgG titers were detectable against only a fraction of the total number of toxins encoded in the genome of the infecting S. pyogenes strain for IVIG-treated and nontreated NSTI cases (Figure 3B). In contrast, IgG titers against all exotoxins encoded by the respective infecting S. pyogenes strain were observed in day 0 plasma specimens collected from both patients in whom the initial STI did not develop into a NSTI (Figure 3B). The antibody profile in plasma samples collected on day 3 of infection indicated that IVIG-untreated patients had increased antibody titers only against some of the toxins encoded in the genomes of the infecting S. pyogenes strains and that IgG titers were still absent against a proportion of exotoxins, including the superantigens SpeI, SpeJ, SpeK, SpeL, and SpeM (Figure 3B). In contrast, the day 3 plasma samples collected from patients with NSTIs who received IVIG treatment displayed detectable antibodies titers against all toxins except SpeG (Figure 3B). These results indicate that IVIG treatment provides patients with NSTIs with a pool of antibodies against a wide array of exotoxins that complement those that may not be induced by natural infection. Indeed, IVIG preparations contained detectable antibody titers against all exotoxins tested in this study (Figure 3C). The antibody profile of plasma samples from patients with nonnecrotizing STIs showed no changes during the course of infection, since all streptococcal exotoxins encoded in the genomes of the infecting S. pyogenes strains were covered by specific antibodies already in day 0 plasma samples (Figure 3B). This may suggest that preexisting antibodies against the repertoire of exotoxins produced by the infecting S. pyogenes strain may prevent the progression of STI to severe NSTI. The opposite could also be the case, and patients lacking antibodies against specific S. pyogenes exotoxins may be at risk for developing NSTI. Patients With NSTIs Lack Antibodies That Neutralize the Effect of S. pyogenes Exotoxins Many of the devastating effects of S. pyogenes NSTI are mediated by the hyperactivation of T cells by streptococcal superantigens, as well as by the tissue damage caused by toxins such as the streptococcal cytolysin SLO, SpeB, and Sic [15]. To determine whether the antibodies present in the plasma of patients with NSTIs are capable to neutralize the effect of these factors, functional assays were performed using plasma samples collected from a representative patient subset. Plasma samples that contained antibodies against specific streptococcal superantigens were highly effective at inhibiting the superantigenicity effect on T-cell proliferation (>90%) of the specific superantigen (Figure 4A and Supplementary Data Set 2). Only in the case of SpeA the inhibition was incomplete (42%–84%). Among plasma lacking antibodies against the specific superantigen, the inhibition never exceeded 12% (Figure 4A). Similar to superantigens, the capacity of a patient’s plasma specimen to suppress the functional effect of SpeB, SLO, and Sic strongly correlated with the presence of antibodies specific for the corresponding toxin (Figure 4A). This indicates that patients developing NSTI did not carry specific antibodies against many of the exotoxins potentially secreted by the infecting bacterial isolate and thus could not inhibit the negative effect of these toxins. In contrast, the antibodies detected in day 0 samples from patients with nonnecrotizing STIs were capable of inhibiting exotoxins encoded by the invading pathogen, and neither change in antibody titers nor in inhibition potential was observed during infection progression (Figure 4A). Notably, plasma specimens from patients who received IVIG treatment were capable of inhibiting the activity of all analyzed exotoxins, with the exception of that of the superantigen SpeA (Figure 4A). To evaluate whether this effect was mediated by toxin-neutralizing antibodies contained in the IVIG preparations, functional assays were performed with IgG-depleted human plasma supplemented with either 1 mg/mL or 5 mg/mL of IVIG. The induction of T-cell proliferation by streptococcal superantigens (SpeA, SpeC, SpeG, SpeH, SpeI, SpeJ, SpeK, SpeL, SpeM, SSA, and SmeZ) was clearly inhibited (>90%) by IVIG even at a concentration of only 20% of the dosage used in clinical settings (1 mg/mL; Figure 4B). SpeA was the only superantigen whose activity was not fully inhibited; however, 73% inhibition was observed when IVIG was administered at the concentration used in the clinic (5 mg/mL; Figure 4B). The activity of the streptococcal toxins SpeB, SLO, and Sic was also effectively neutralized by the clinically relevant concentration of IVIG (Figure 4B). This demonstrates that IVIG preparations contain antibodies against 14 streptococcal exotoxins that, when used in the clinically appropriate concentration, are able to completely inhibit the function of 13 of them, leaving only SpeA as partially inhibited. Figure 4. View largeDownload slide Exotoxin neutralizing capacity of plasma and intravenous immunoglobulin (IVIG) preparations. A, Antibody titers against streptococcal exotoxins in patient plasma and their capability to inhibit the exotoxins function. Antibody titers were determined against 14 streptococcal exotoxins identified in the corresponding Streptococcus pyogenes isolates from patients with necrotizing soft-tissue infection (NSTI) or nonnecrotizing STI, using plasma samples collected upon admission to the hospital (day 0) and 3 days after admission (day 3). Toxin activities were determined by a T-cell proliferation assay (for superantigens), an LL-37 protection assay (for Sic), an azocasein assay (for SpeB), or an erythrocyte cytotoxicity assay (for streptolysin O [SLO]) after incubation in the corresponding patient plasma sample. Percentage inhibition was calculated relative to the 100% toxin activity determined in immunoglobulin G (IgG)–depleted plasma and is indicated in the respective boxes. Red, exotoxin gene is present in the bacterial genome but no significant antibody titer is in the corresponding patient plasma sample; blue, gene is present in the bacterial genome and an antibody titer is present in plasma; gray, gene is absent in the bacterial genome but antibody titers are detectable in plasma; white, gene is absent in the bacterial genome and antibody titers are undetectable in plasma. B, Inhibitory capacity of IVIG preparations of 14 streptococcal exotoxins. Toxin activities were determined by a T-cell proliferation assay (for superantigens), an azocasein assay (for SpeB), an LL-37 protection assay (for Sic), or an erythrocyte cytotoxicity assay (for SLO) after incubation in IgG-depleted human plasma containing 1 mg/mL IVIG (gray bars) or 5 mg/mL IVIG (black bars). Inhibition is calculated relative to the 100% activity determined in IgG-depleted plasma without IVIG. Data represent arithmetic means ± standard deviations of two individual experiments. Figure 4. View largeDownload slide Exotoxin neutralizing capacity of plasma and intravenous immunoglobulin (IVIG) preparations. A, Antibody titers against streptococcal exotoxins in patient plasma and their capability to inhibit the exotoxins function. Antibody titers were determined against 14 streptococcal exotoxins identified in the corresponding Streptococcus pyogenes isolates from patients with necrotizing soft-tissue infection (NSTI) or nonnecrotizing STI, using plasma samples collected upon admission to the hospital (day 0) and 3 days after admission (day 3). Toxin activities were determined by a T-cell proliferation assay (for superantigens), an LL-37 protection assay (for Sic), an azocasein assay (for SpeB), or an erythrocyte cytotoxicity assay (for streptolysin O [SLO]) after incubation in the corresponding patient plasma sample. Percentage inhibition was calculated relative to the 100% toxin activity determined in immunoglobulin G (IgG)–depleted plasma and is indicated in the respective boxes. Red, exotoxin gene is present in the bacterial genome but no significant antibody titer is in the corresponding patient plasma sample; blue, gene is present in the bacterial genome and an antibody titer is present in plasma; gray, gene is absent in the bacterial genome but antibody titers are detectable in plasma; white, gene is absent in the bacterial genome and antibody titers are undetectable in plasma. B, Inhibitory capacity of IVIG preparations of 14 streptococcal exotoxins. Toxin activities were determined by a T-cell proliferation assay (for superantigens), an azocasein assay (for SpeB), an LL-37 protection assay (for Sic), or an erythrocyte cytotoxicity assay (for SLO) after incubation in IgG-depleted human plasma containing 1 mg/mL IVIG (gray bars) or 5 mg/mL IVIG (black bars). Inhibition is calculated relative to the 100% activity determined in IgG-depleted plasma without IVIG. Data represent arithmetic means ± standard deviations of two individual experiments. DISCUSSION The development of a NSTI is characterized by a complex interplay between the host immune system and the invading pathogen that produces a wide array of virulence factors to evade and manipulate the immune response. Owing to the crucial role of antibodies in host defense against S. pyogenes [9], the present study investigated the role of pathogen-specific antibodies in the development and progression of S. pyogenes NSTI. Although the microbiologic background of NSTI is considerably diverse [1], we confirmed the previously reported dominating role of S. pyogenes as an etiologic agent, especially those of emm type emm1 [1, 16–19]. The clinical isolates in this study also included emm types more rarely observed in invasive infections such as emm77 and emm63 [18, 20]. Although S. pyogenes is a frequent cause of skin infections, the reason why in particular patients, these infections progress into severe NSTI remains unclear. The results of this study indicate that the absence of preexisting antibody titers against pathogen-specific determinants in patient sera during the early stage of tissue infection may represent a risk factor for the development of a NSTI. This was substantiated by the observation that opsonophagocytic killing rates of the infecting S. pyogenes strain by the corresponding patient plasma specimen collected on day 0 were significantly lower in patients with NSTIs than in nonnecrotizing STI control cases. These observations suggest a direct link between an individual’s serologic profile and their risk of progression into severe NSTI. We also provide evidence that deficiency of pathogen-specific opsonic antibodies in patients with NSTIs can be compensated, at least to some extent, by the administration of IVIG. Streptococcal NSTI is frequently treated with IVIG, a pool of concentrated human serum antibodies [19]. Even though few reports suggested that IVIG contains protective antibody titers against S. pyogenes, its efficacy in the NSTI scenario remains ambiguous [21]. As many of the adverse effects associated with the progression of NSTI are mediated by the activity of superantigen and toxins [22–24], the presence of antibodies capable of neutralizing the activity of these bacterial factors was also investigated here. Interestingly, no difference in the exotoxin gene profile between nonnecrotizing STI and NSTI bacterial isolates was observed, confirming that differences in virulence factor profiles may explain the invasiveness and outcome of S. pyogenes infections only to a limited extent [25]. IVIG have been so far only investigated for the presence of antibodies against the superantigens SpeA, SpeC, and SpeG [26]. We could identify significant antibody titers against 14 additional toxins potentially involved in the development of NSTI. The combined effect of enhancing opsonophagocytic killing and neutralizing the full array of bacterial superantigens and toxins highlights the potential benefit of using IVIG as adjunctive therapy for limiting the progression of NSTI. However, IVIG therapy seems to be of benefit only if administered during the very early stage of NSTI, since administration of IVIG at later stages of infection will not invert the progression of an already established NSTI. This may explain the results obtained from clinical studies, where no significant effect of IVIG treatment on the outcome and mortality of NSTI was observed [21, 27]. The antibody profile observed in the non–IVIG-treated NSTI cases clearly discriminates between bacterial factors able to induce a robust antigen-specific antibody response, like SpeA, SpeC, SpeG, SpeH, or SmeZ, and nonimmunogenic toxins, such as SpeI, SpeJ, SpeK, SpeL, and SpeM, which failed to induce an antibody response. These differences in antibody levels during the acute phase of infection probably reflect a preexisting memory B- and T-cell response only against a set of bacterial antigens that can be activated very fast after a second exposure to the pathogen [28]. We can, therefore, hypothesize that the subset of analyzed bacterial toxins, which does not induce a specific antibody response, is either not expressed during the infection process and thereby not involved in the NSTI progression or that the patient has never been confronted with a pathogen producing these factors and therefore lacks specific memory responses. Although S. pyogenes strains carrying speL, speM, and speK have been more regularly isolated from cases of acute rheumatic fever [29, 30], a direct association of these superantigens with invasive streptococcal infections like NSTI is not known and should be addressed in future studies. Antibodies can mediate protection against streptococcal toxins by neutralizing the activity of a specific toxin. Using functional assays, we demonstrated the efficiency of IVIG, used at a clinically relevant dosage, in neutralizing 13 streptococcal exotoxins. The observation that the potent superantigen SpeA cannot be blocked completely by antibodies is in accordance with previous studies [31] and is discussed to be linked to its binding specificity to the major histocompatibility complex class II receptor α chain, which discriminates SpeA from all other streptococcal superantigens [32]. In summary, the results presented here are of major clinical relevance. Even though the incidence of NSTI is low, increasing numbers of affected cases have been observed [33]. The dramatic manifestation, unclear origin, and complicated diagnosis of this life-threatening infection make the identification of risk factors a major challenge. The study presented here identified a state of serologic susceptibility potentially linked to the irreversible progression of an initial tissue infection to a severe NSTI. This interpretation suggests that a quick intervention with a high dose of IVIG during the early stage is potentially able to interfere with infection progression and, in that way, not only positively influences the efficiency of other antimicrobial treatment strategies but also decreases the risk of side effects, thereby increasing the chance of survival among affected patients. STUDY GROUP MEMBERS Members of the INFECT study group are as follows: Anna Norrby-Teglund, Mattias Svensson, Anders Rosén, Ylva Karlsson, Martin B Madsen, Steinar Skrede, Oddvar Oppegaard, Torbjørn Nedrebø, and Eva Medina. Affiliations for all study group members are specified in the Supplementary Materials. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Acknowledgments.  We thank the hospital teams in Denmark, Sweden, and Norway that were involved in INFECT project, for their help with patient inclusion, collection of blood specimens, and entering of clinical information into the database; Dr Giuseppe Gulotta, for providing the modified pQE30 vector containing the TEV cleavage site (pQE30-TEV); Dr René Bergmann, for his technical help and fruitful discussions; and Dr Eva Medina and Dr Oliver Goldmann, for their critical review of manuscript and scientific input. Author contributions. A. I. designed the study. A. B. and A. I. performed the experiments and analyzed the data. The INFECT Study Group contributed to clinical study design, study planning, patient recruitment, patient sample collection, typing, and patient data analyses. A. B., A. I., and D. H. P. wrote the manuscript. All authors read and approved the final version. Financial support. This work was supported by the European Union’s Seventh Framework Program (grant 305340). Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Presented in part: 20th Lancefield International Symposium on Streptococci and Streptococcal Disease, 16–20 October 2017, Fiji. References 1. Misiakos EP , Bagias G , Patapis P , Sotiropoulos D , Kanavidis P , Machairas A . Current concepts in the management of necrotizing fasciitis . Front Surg 2014 ; 1 – 36 . 2. Bisno AL , Stevens DL . 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The Journal of Infectious DiseasesOxford University Press

Published: Mar 3, 2018

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